Nucleoside analogs and uses in treating disease

ABSTRACT

The invention relates to pharmaceutical compositions of nucleoside dimers containing an L-sugar in at least one of the nucleosides.

This Application is a continuation-in-part from U.S. patent applicationSer. No. 08/531,875, filed on Sep. 21, 1995 now U.S. Pat. No. 6,025,335.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to novel nucleosides and dinucleoside dimers andderivatives of these compounds, including, L-deoxyribofuranosylnucleoside phosphodiester dimers in which the sugar moiety of at leastone of the nucleosides has an L-configuration. These compounds arehighly effective in the treatment of various diseases. They may be usedto treat parasitic infections. They may also be used to treat bacterial,viral, and fungal infections, and may also be used to treat cancer.

2. Prior Art

Modified nucleoside analogs are an important class of antineoplastic andantiviral drugs. The present application discloses novel compounds forof this type for use in the treatment of parasitic infections. Due tothe extraordinary morbidity and mortality associated with parasiticinfections, related research has intensified during the past decade in adesperate search for effective treatments. A safe and effective vaccinestill does not exist. Instead, victims must depend upon chemotherapy.

Furthermore, these modified nucleoside analogs may be used to treatbacterial infections, fungal infections, viral infections, and cancer.

These chemotherapeutic agents can be classified into two groups: thosethat act post-translationally, and those that act by interfering withnucleic acid synthesis.

Most drugs are in the first group, which means that they exert theirtherapeutic effect by interfering with a cell's protein synthesis, andhence its metabolism (rather than its nucleic acid synthesis). Examplesof drugs in this group include: the antifolate compounds (which inhibitdihyrdofolate reductase), and sulfonamide drugs (which inhibitdihydropteroate synthetase. Yet these drugs have serious drawbacks. Forexample, the protozoan responsible for malaria very quickly developsresistance to these drugs. The reason is that, since resistance occursthrough adaptive mutations in successive generations of the parasite, aone or two point mutation is often sufficient to confer resistance.Bacterial, viral, and fungal infections are frequently also susceptibleto these types of resistance mutations.

The second group of compounds includes the nucleic acid intercalatorssuch as acridines, phenanthrenes and quinolines. These intercalatorspartially mimic the biochemical activity of nucleic acids, and thereforeare incorporated into a cell's nucleic acid (DNA and RNA), though onceincorporated, do not allow further nucleic acid synthesis, hence theireffectiveness. At the same time, these intercalators interfere with hostnucleic acid synthesis as well, and thus give rise to toxic sideeffects. Because of the potential for toxic side effects, these drugscan quite often be given only in very small doses. Once again, aresistance pattern may develop. For example, some protozoans are knownto develop “cross-resistance,” which means that the parasites developresistance to other classes of drugs even though they were exposed on adifferent class of drug.

Indeed, all of the currently known drugs or drug candidates utilizingthe delivery of cytotoxic pyrimidine or purine biosynthesis inhibitorsto invading cells are extremely toxic. Therefore, while drugs of thistype—i.e., those that interfere with nucleic acid synthesis-areeffective, they lack selectivity. It is this latter parameter that mustbe maximized in the development of a safe and effective drug. In otherwords, such a drug would target host tissues that are infected, orcancerous, yet leave the host tissue unchanged.

Recent advances in our understanding of the biochemistry of parasitecells serves as a valuable example regarding the design of effectivetherapies. One investigator (H. Ginsburg, Biochem. Pharmacol. 48,1847-1856 (1994)) observed that normal and parasite-infectederythrocytes exhibit significant differences with respect to purine andpyrimidine metabolism in single enzymes, as well as in whole branches ofrelated pathways. The parasite satisfies all of its purine requirementsthrough scavenger pathways; meanwhile, the host cell lacks the enzymesnecessary to exploit this pathway, and so therefore must meet itspyrimidine requirements largely through de novo synthesis. Put anotherway, the parasite is more efficient than normal or host cells since itcan synthesize the nucleic acid building blocks.

Other investigators (G. Beaton, D. Pellinqer, W. S. Marshall & M. H.Caruthers, In: Oligonucleotides and Analogues: A Practical Approach, F.Eckstein Ed., IRL Press, Oxford, 109-136 (1991)) have established that amalaria-infected erythrocyte is capable of effectively transporting thenon-naturally occurring “L-nucleosides” (in contrast to the“D-nucleosides” which are the naturally occurring form) for use innucleic acid synthesis. Yet, normal mammalian cells are nonpermeable tothis class of compounds, which suggests that the L-nucleosides arenon-toxic to normal mammalian or host cells. Thus, derivatives of thesecompounds may be used as highly selective drugs against parasiteinfection, or against any other type of cell or organism utilizing theL-nucleosides. The chemical modification of the L-nucleosides consistsgenerally of modifying the nucleosides so that they are still recognizedby the invading cell or organism's nucleic acid synthetic machinery, andtherefore incorporated into a nucleic acid chain, but yet once thisincorporation occurs, no further synthesis will take place.

Currently, there are no therapeutic compounds in use that are based ondimers of these nucleoside analogs. While dimers of the naturallyoccurring D-deoxyribofuranosyl nucleosides are well known, dimers inwhich one or both nucleosides are of the unnatural L-configuration aremuch less known, and their use in therapy of neoplastic and viraldiseases is unknown.

In the synthesis of DNA-related oligomers, types of nucleoside dimersare synthesized as part of the overall process. These dimers usuallyinclude bases from naturally occurring DNA or RNA sequences. There ismuch known in the art about nucleoside monophosphate dimers. Many ofthese compounds have been synthesized and are available commercially.

However, these dimers are made from nucleosides containing a sugarmoiety in D-configuration.

Reese, C. B., Tetrahedron 34 (1978) 3143 describes the synthesis offully-protected dinucleoside monophosphates by means of thephosphotriester approach.

Littauer, U. Z., and Soreg, H. (1982) in The Enzymes, Vol. XV, AcademicPress, NY, p. 517 is a standard reference which describes the enzymaticsynthesis of dinucleotides.

Heikkilö, J., Stridh, S., Oberg, B. and Chattopodhyaya, J., Acta Chem.Scand. B 39 (1985) 657-669, provides an example of the methodology usedin the synthesis of a variety of ApG nucleoside phosphate dimers.Included are references and methods for synthesis of 3′→5′ phosphatesand 2′→5′ phosphates by solution phase chemistry.

Gait, M., “Oligonucleotide Synthesis”, IRL Press, Ltd., Oxford, England,1984, is a general reference and a useful overview for oligonucleotidesynthesis. The methods are applicable to synthesis of dimers, both bysolution phase and solid phase methods. Both phosphitetriester andphosphotriester methods of coupling nucleosides are described. The solidphase method is useful for synthesizing dimers.

Gulyawa, V. and Holy, A., Coll. Czec. Chem. Commun 44 613 (1979),describe the enzymatic synthesis of a series of dimers by reaction of2′,-3′ cyclic phosphate donors with ribonucleoside acceptors. Thereaction was catalyzed by non-specific RNases. The donors arephosphorylated in the 5′-position, yielding the following compounds:donor nucleoside-(3′→5′) acceptor nucleoside. Dimers were made withacceptors, β-L-cytidine, β-L-adenosine, and 9(α-L-lyxofuranosyl)adenine. Also, a large number of dimers with D-nucleosides in theacceptor 5′-position were made.

Holy, A., Sorm, F., Collect. Czech. Chem. Commun., 34, 3383 (1969),describe an enzymatic synthesis of β-D-guanylyl-(3′→5′)-β-L-adenosineand β-D-guanylyl-(3′→5′)-β-L-cytidine.

Schirmeister, H. and Pfleiderer, W., Helv. Chim. Acta 77, 10 (1994),describe trimer synthesis and intermediate dimers, all fromβ-D-nucleosides. They used the phosphoramidite method which gave goodyields.

Thus, dimers with L-deoxyribofuranosyl moieties in any position are new,as are dimers with L-ribofuranosyl moieties bonded to the 3′-position ofthe phosphate internucleotide bond.

Modified nucleoside analogues represent an important class of compoundsin the available arsenal of antineoplastic and antiviral drugs. Theanticancer agents 5-fluorodeoxyuridine (floxuridine), cytarabine anddeoxycoformycin and the antiviral drugs 3′azidodeoxythymidine (AZT),dideoxycytidine (ddC), dideoxyinosine (ddl), acyclovir,5-iododeoxyuridine (idoxuridine) fludarabine phosphate and vidarabine(adenine arabinoside/ara A) are representative of this class ofmonomeric nucleoside-derived compounds which are used therapeutically.

More recently, “antisense” oligonucleotide analogues with modified basesand/or phosphodiester backbones have been actively pursued as antiviraland antitumor agents. While no clinically approved drug has yet emergedfrom this class of compounds, it remains a very active field ofresearch. Recently, antipodal L-sugar-based nucleosides also have foundapplication as potent antiviral agents because they can inhibit viralenzymes without affecting mammalian enzymes, resulting in agents thathave selective antiviral activity without concomitant mammaliancytotoxicity. Most naturally occurring nucleosides have theD-configuration in the sugar moiety. While the chemical properties ofL-nucleosides are similar to those of their β-D-enantiomers, theyexhibit very different biological profiles in mammalian cells and do notinterfere with the transport of normal D-nucleosides. For example,β-L-uridine is not phosphorylated at the 5′-position by human prostatephosphotransferase, which readily phosphorylates the enantiomericβ-D-uridine. Apparently, L-nucleosides are not substrates for normalhuman cell kinases, but they may be phosphorylated by viral and cancercell enzymes, allowing their use for the design of selective antiviraland anticancer drugs. Oligonucleotides based on L-nucleosides have beenstudied previously. Octamers derived from α- and β-L-thymidine werefound resistant to fungal nucleases and calf spleen phosphodiesterase,which readily degrades the corresponding β-D-oligonucleotide. Fujimory,et al., S.

Fujimory, K. Shudo, Y. Hashimoto, J. Am. Chem. Soc., 112, 7436, haveshown that enantiomeric poly-α-DNA recognizes complementary RNA but notcomplementary DNA. This principle has been used in the design ofnuclease-resistant antisense oligonucleotides for potential therapeuticapplications.

Thus, L-nucleoside-based compounds have potential as drugs againstneoplastic, fungal, and viral diseases, as well as against parasiticinfections.

While L-sugar-derived nucleosides and their oligonucleotides have beenwidely evaluated for such activities, little is known regarding thebiological activities of shorter oligomers such as dimers obtained byL-nucleoside substitution.

This invention comprises novel L-nucleoside-derived therapeuticantitumor, antiviral, antibacterial, antifungal, and antiparasiticagents. Novel L-nucleoside-derived dinucleoside monophosphates, based onL-α-5-fluoro-2′-deoxyuridine showed a remarkably high potency activityprofile in in vitro anti-cancer assays, with indications of uniquemechanisms of action, including inhibition of telomerase. Therefore, theL-nucleosides can serve as building blocks for new drugs with thespecial advantage of low toxicity.

SUMMARY OF THE INVENTION

A further embodiment of the present invention is the administration of atherapeutically effective amount of the compounds of the presentinvention for the treatment of cancer, viral infections, parasiticinfections, fungal infections, and bacterial infections.

Other and further objects, features and advantages will be apparent fromthe following description of the present preferred embodiments of theinvention given for the purposes of disclosure when taken in conjunctionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of examples of the dinucleotidedimers of the present invention.

FIG. 2 is a schematic representation of part of the synthesis scheme ofcompound N⁴benzoyl-2′-deoxy-alpha-Lcytidine, an intermediate in thesynthesis of the dimers.

FIG. 3 is a schematic representation of part of the synthesis scheme forcompounds of the present invention (2′-deoxy-alpha-L-5-fluoriuridenecompound).

FIG. 4 is a schematic representation of the synthesis of a specificseries of alpha-beta or beta-alpha dimers.

FIG. 5 is a schematic representation of the synthesis of an α-L-5fluorouracil-β-D-5 fluorouracil.

FIG. 6 is a schematic representation of an alternative pathway for thesynthesis of α-L-5 fluorouracil-β-D-5 fluorouracil.

FIG. 7 is a schematic representation for the synthesis of dimercompounds, L-150.

FIG. 8 is a schematic representation of a synthesis pathway for a dimercompound. L-151.

FIG. 9 is a schematic representation of the synthesis pathway of α-L-dU,Cordycepin dimer, L-152.

FIG. 10 is a schematic representation of the synthesis of β-L-dC,Cordycepin dimer, L-153.

FIG. 11 is a schematic representation of the synthesis pathway ofα-L-dC, Cordycepin dimer, L-154.

FIG. 12 is a schematic representation of the synthesis pathway of α-dA,Cordycepin dimer, L-155.

FIG. 13 is a schematic representation of the synthesis of -L-β-dA,β-D-dA dimer, L-210.

FIGS. 14A and 14B are schematic representations of dinucleosidephosphate dimers containing alternative backbones.

FIG. 14A shows the methoxyphosphotriesters, methylphosphonates,phosphorodithioates, and silylethers. FIG. 14B shows the sulfonates,ethylenedioxyesters, and phosphorotioates that can be used as thebackbone.

FIGS. 15A, 15B, 15C, 15D, and 15E are schematic representations ofdinucleoside phosphate dimers used in the examples.

FIG. 15A shows a schematic representation of the dinucleotide dimers:GCI-1077, GCI-1079, GCI-1085, GCI-1086, FUdR, L-145, L-147, L-146 andL-144.

FIG. 15B shows a schematic representation of the dimers: L-133, L-138,GCI-1007, GCI-1018, G-1027, GCI-1030, GCI-1032, GCI-1033, GCI-1034,GCI-1036, GCI-1037, GCI-1066, GCI-1069, and GCI-1070.

FIG. 15C shows a schematic representation of the dimers: L-113,L-117thio, L-117, L-120, L-125, L-122, L-124, and L-128.

FIG. 15D shows a schematic representation of the dimers: L-101, L-103,L-103thio, L-103CN, L-107, L-109, L-110, L-111, and L-112.

FIG. 15E shows a schematic representation of dimers: B01, B02, B03, B04,B05, B06, and B07.

Certain features of the invention may be exaggerated in scale or shownin schematic form in accordance with the customary practices in thebiochemical arts.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is readily apparent to one skilled in the art that varioussubstitutions and modifications may be made to the invention disclosedin this Application without departing from the scope and spirit of theinvention.

The term “dimers” as used herein is defined by the structures shown inFIG. 1. These compounds are L-nucleoside-derived dinucleosidemonophosphates. The B. and B₂ units will consist of either a β-D, a β-Lor an α-L nucleoside and at least one of B₁ or B₂ will be β-L or α-L. R₁and R₂ will be the pyrimidine bases cytosine, thymine, uracil, or5-fluorouridine (5-FUdR) other 5-halo compounds, or the purine bases,adenosine, guanosine or inosine. As can be seen in FIG. 1, the dimerscan be bound byvarious linkages. Permissible linkages include 5′→3′,3′→5′, 3′→3′, 5′→5′, 2′→3′, 3′→2′, 2′→2′, 2′→5′, 5′→2′, or any otherstereochemically permissible linkage. The sugar part of the nucleosidemay be fully oxygenated, or may be in the deoxy or dideoxy form.

Specific antidisease compounds which are useful in the present inventioninclude3′-O-(α-L-5-fluoro-2′-deoxyuridinyl)-β-D-5-fluoro-2′-deoxyuridine,(L-102),3′-O-(β-D-5-fluoro-2′-deoxyuridinyl)-α-L-5-fluoro-2′-deoxyuridine,(L-103), 3′-O-(β-D-5-fluoro-2′-deoxyuridinyl)-α-L-2′-deoxyuridine,(L-107),3′-O-(α-L-5-fluoro-2′-deoxyuridinyl)-α-L-5-fluoro-2′-deoxyuridine,(L-108),3′-O-(β-L-5-fluoro-2′-deoxyuridinyl)-β-L-5-fluoro-2′-deoxyuridine,(L-109),3′-O-(β-D-5-fluoro-2′-deoxyuridinyl)-β-L-5-fluoro-2′-deoxyuridine,(L-110), 3′-O-(β-D-5-fluoro-2′-deoxyuridinyl)-α-L-2′-deoxycytidine,(L-111), 3′-O-(β-D-5-fluoro-2′-deoxyuridinyl)-2′-deoxy-β-L-cytidine(L-113), 3′-O-(2′-deoxy-β-L-cytidinyl)-3-D-5-fluoro-2′-deoxyuridine(L-114), 3′-O-(2′-deoxy-α-L-cytidinyl)-β-D-5-fluoro-2′-deoxyuridine(L-115), 3′-O-(β-D-5-fluoro-2′-deoxyuridinyl)-β-L-2′-deoxyuridine(L-117),3′-O-(β-L-5-fluoro-2′-deoxyuridinyl)-α-L-5-fluoro-2′-deoxyuridine (L-119), 3′-O-(β-D-5-fluoro-2′-deoxyuridinyl)-α-L-5-fluoro-2′-deoxyuridine(3′, 3′) (L-122), 3′-O-(3′-deoxy-β-D-adenosinyl)-β-L-2′-deoxyuridine(L-150), 3′-O-(3′-deoxy-β-D-adenosinyl)-β-L-2′-deoxyadenosine (L-151),3′-O-(3′-deoxy-β-D-adenosinyl)-α-L-2′-deoxyuridine (L-152),3′-O-(3′-deoxy-β-D-adenosinyl)-β-L-2′-deoxycytidine (L-153),3′-O-(3′-deoxy-β-D-adenosinyl)-α-L-2′-deoxycytidine (L-154),3′-O-(3′-deoxy-β-D-adenosinyl)-β-L-2′-deoxyadenosine (L-155),3′-O-(2′-deoxy-β-D-adenosinyl)-β-L-2′-deoxyadenosine (L-210). In thecurrently preferred embodiment,3′-O-(β-D-5-fluoro-2′-deoxyuridinyl)-α-L-5-fluoro-2′-deoxyuridine,(L-103) is used.

The term “internucleotide binding agent” or “IBA” means the backbonebinding which links the nucleosides together. Although one skilled inthe art will readily recognize a variety of other backbones areavailable and useful in the present invention. For example, see FIG. 1,where methoxy phosphotriesters, methylphosphonates, phosphorodithioates,phosphorothioates, silyl ethers, sulphonates and ethylenedioxy ethersare shown. Although shown schematically 3′-5′ the IBA's can be used tolink the sugars 5′→3′, 3′→5′, 3′→3′, 5′→5′, 2′→3′, 3′→2′, 2′→2′, 2′→5′,5′→2′, or any other stereochemically permissible linkages. In thepreferred embodiment, the IBA of the compounds is either phosphodiesteror phosphorothioate. The term “antidisease” as used herein refers to anyof the activities of the compounds of the present invention to affect adisease state, including antitumor, antineoplastic, anticancer,antiparasitic and antiviral activity.

A compound or composition is said to be “pharmacologically acceptable”if its administration can be tolerated by a recipient mammal. Such anagent is said to be administered in a “therapeutically effective amount”if the amount administered is physiologically significant. An agent isphysiologically significant if its presence results in technical changein the physiology of a recipient mammal. For example, in the treatmentof cancer or neoplastic disease, a compound which inhibits the tumorgrowth or decreases the size of the tumor would be therapeuticallyeffective; whereas in the treatment of a viral disease, an agent whichslows the progression of the disease or completely treats the disease,would be considered therapeutically effective.

Dosage and Formulation

The antidisease compounds (active ingredients) of this invention can beformulated and administered to inhibit a variety of disease states(including tumors, neoplasty, cancer, bacterial, fungal, parasitic andviral diseases) by any means that produces contact of the activeingredient with the agent's site of action in the body of a mammal. Theycan be administered by any conventional means available for use inconjunction with pharmaceuticals, either as individual therapeuticactive ingredients or in a combination of therapeutic activeingredients. They can be administered alone, but are generallyadministered with a pharmaceutical carrier selected on the basis of thechosen route of administration and standard pharmaceutical practice.

The dosages given as examples herein are the dosages usually used intreating tumors, neoplasty and cancer. Dosages for antiparasitic andantiviral applications will, in general, be 10-50% of the dosages foranticancer applications.

The dosage administered will be a therapeutically effective amount ofactive ingredient and will, of course, vary depending upon known factorssuch as the pharmacodynamic characteristics of the particular activeingredient and its mode and route of administration; age, sex, healthand weight of the recipient; nature and extent of symptoms; kind ofconcurrent treatment, frequency of treatment and the effect desired.Usually a daily dosage (therapeutic effective amount) of activeingredient can be about 5 to 400 milligrams per kilogram of body weight.Ordinarily, 10 to 200, and preferably 10 to 50, milligrams per kilogramper day given in divided doses 2 to 4 times a day or in sustainedrelease form is effective to obtain desired results.

Dosage forms (compositions) suitable for internal administration containfrom about 1.0 to about 500 milligrams of active ingredient per unit. Inthese pharmaceutical compositions, the active ingredient will ordinarilybe present in an amount of about 0.05-95% by weight based on the totalweight of the composition.

The active ingredient can be administered orally in solid dosage formssuch as capsules, tablets and powders, or in liquid dosage forms such aselixirs, syrups, emulsions and suspensions. The active ingredient canalso be formulated for administration parenterally by injection, rapidinfusion, nasopharyngeal absorption or dermoabsorption. The agent may beadministered intramuscularly, intravenously, or as a suppository.

Gelatin capsules contain the active ingredient and powdered carrierssuch as lactose, sucrose, mannitol, starch, cellulose derivatives,magnesium stearate, stearic acid, and the like. Similar diluents can beused to make compressed tablets. Both tablets and capsules can bemanufactured as sustained release products to provide for continuousrelease of medication over a period of hours. Compressed tablets can besugar coated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose),and related sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solutions for parenteral administration contain preferably a watersoluble salt of the active ingredient, suitable stabilizing agents and,if necessary, buffer substances. Antioxidizing agents such as sodiumbisulfate, sodium sulfite or ascorbic acid, either alone or combined,are suitable stabilizing agents. Also used are citric acid and its saltsand sodium EDTA. In addition, parenteral solutions can containpreservatives such as benzalkonium chloride, methyl- or propyl-parabenand chlorobutanol. Suitable pharmaceutical carriers are described inRemington's Pharmaceutical Sciences, a standard reference text in thisfield.

Additionally, standard pharmaceutical methods can be employed to controlthe duration of action. These are well known in the art and includecontrol release preparations and can include appropriate macromolecules,for example polymers, polyesters, polyaminoacids, polyvinyl, pyrolidone,ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose orprotamine sulfate. The concentration of macromolecules as well as themethods of incorporation can be adjusted in order to control release.Additionally, the agent can be incorporated into particles of polymericmaterials such as polyesters, polyaminoacids, hydrogels, poly (lacticacid) or ethylenevinylacetate copolymers. In addition to beingincorporated, these agents can also be used to trap the compound inmicrocapsules.

Useful pharmaceutical dosage forms for administration of the compoundsof this invention can be illustrated as follows.

Capsules:

Capsules are prepared by filling standard two-piece hard gelatincapsulates each with 100 milligram of powdered active ingredient, 175milligrams of lactose, 24 milligrams of talc and 6 milligrams magnesiumstearate.

Soft Gelatin Capsules:

A mixture of active ingredient in soybean oil is prepared and injectedby means of a positive displacement pump into gelatin to form softgelatin capsules containing 100 milligrams of the active ingredient. Thecapsules are then washed and dried.

Tablets: Tablets are prepared by conventional procedures so that thedosage unit is 100 milligrams of active ingredient. 0.2 milligrams ofcolloidal silicon dioxide, 5 milligrams of magnesium stearate, 275milligrams of microcrystalline cellulose, 11 milligrams of cornstarchand 98.8 milligrams of lactose. Appropriate coatings may be applied toincrease palatability or to delay absorption.

Injectable:

A parenteral composition suitable for administration by injection isprepared by stirring 1.5% by weight of active ingredients in 10% byvolume propylene glycol and water. The solution is made isotonic withsodium chloride and sterilized.

Suspension:

An aqueous suspension is prepared for oral administration so that each 5millimeters contain 100 milligrams of finely divided active ingredient,200 milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodiumbenzoate, 1.0 grams of sorbitol solution U.S.P. and 0.025 millimeters ofvanillin.

SUMMARY OF COMPOUNDS SYNTHESIZED

The following examples are offered by way of illustration and are notintended to limit the invention in any manner. The nucleosides anddimers may incorporate any stereochemcially permissible linkage na mayinclude various deoxy forms of the sugar rings. The syntheticnucleosides and dimers described in the examples can include any of thesubstitutions discussed earlier. The backbone and base modifying groupscan be added. Various substitutions will enhance the affinity, thechemical stability and the cellular uptake properties of the specificdimers treatments.

EXAMPLE I Synthesis of 2′-deoxy-α-L-5-flourouridine

While β-D-5-fluoro-deoxyuridine is commercially available, theα-L-isomer 2′-deoxy-α-L-5-fluorouridine is not, and this component ofthe dimers was synthesized from L-arabinose.

1-(2′, 3′, 5′-tri-O-benzoyl-α-L-arabinofuranosyl)-5-fluorouracil (3)

To a mixture of 5-fluorouracil (4.01 g, 30.87 mmol) and compound 2(15.57 g, 30.87 mmol) in anhydrous MeCN were successively added HMDS(5.20 ml, 24.69 mmol), ClSiMe₃ (3.10 ml, 24.69 mmol), and SnCl₄ (4.30ml, 37.04 mmol). The resulting clear solution was refluxed for one hour.Then the solvent was evaporated and the residue was dissolved in EtOAc(750 ml), washed with H₂O, and saturated NaHCO₃ solution. The EtOAclayer was dried over sodium sulfate, filtered and evaporated to give thecrude product. This crude product was purified on a silica gel columnusing 40-50% EtOAc/petroleum ether to give pure 3 (11.7 g, 66.0% yield)as a white foam.

NMR: (CDCl₃) δ=4.65 (dd, 1H), 4.78 (dd, 1H), 4.97 (dd, 1H. 5.75-5.88 (2t, 2H), 6.27 (d, 1H), 7.36-7.62 and 8.00-8.10 (m, 5H), 8.94 (d, 1H).

1-α-L-arabinofuranosyl-5-fluorouracil (4)

To a solution of compound 3 (11.7 g, 20.37 mmol) in MeOH (300 ml), NaOMe(4.2 ml of a methanolic 25% w/v solution) was added and the solution wasstirred until the reaction was complete. The solvent was then evaporatedand the residue was dissolved in H₂O (200 ml), washed with ether andneutralized with Dowex 50 ion exchange resin. After filtration of theresin, the aqueous solution was evaporated to give compound 4 (4.92 g,92% yield) as a white foam.

NMR: (DMSO-d₆) δ=3.48 (m, 2H), 3.934.00 (2 t, 2H), 4.16 (q, 1H), 5.69(dd, 1H), 8.03 (d, 1H).

1-[3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-α-L-arabinofuranosyl]-5-fluorouracil(5)

To a stirred suspension of 4 (6.43 g, 24.52 mmol) in pyridine (200 ml)was added 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (10.3 ml, 29.43mmol). This was stirred at room temperature until the reaction wascomplete (5 hours). The solvent was evaporated to a residue which wasdissolved in EtOAC and washed successively with H₂O, 5% HCl, H₂O,saturated NaHCO₃, and brine. After drying the EtOAc portion over Na₂SO₄,the solution was filtered and evaporated to give the crude product 5which was used in the next step without further purification.

1-[2′-O-phenoxythiocarbonyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-α-L-arabinofuraosyl]-5-fluorouracil(6)

To a solution of 5 (24.52 mmol) in anhydrous MeCN (300 ml) were added4-dimethylaminopyridine (DMAP) (5.80 g, 47.58 mmol), andphenylchlorothionoformate (3.85 ml, 26.98 mmol). The solution wasstirred at room temperature for 24 hours. Then, the solvent wasevaporated to a residue which was dissolved in EtOAc and washedsuccessively with H₂O, 5% HCl, H₂O, saturated NaHCO₃, and brine. Afterdrying the EtOAc portion over Na₂SO₄, the solution was filtered andevaporated to an oil. The oil was purified on a silica gel column using30% EtOAc/petroleum ether to produce pure 6 (8.9 g, 56.7% yield) as ayellow foam.

NMR: (CDCl₃) δ=4.02 (m, 2H), 4.32 (m, 1H), 4.76 (dd, 1H), 6.10 (dd, 1H),6.18 (dd, 1H), 7.07-7.48 (m, 6H), 8.41 (br s, 1H).

3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-α-L-2′-deoxy-5-fluorouridine(7)

To a solution of 6 (8.92 g, 13.91 mmol), in dry toluene (300 ml) wasadded AIBN (0.46 g, 2.78 mmol) followed by Bu₃SnH (20.0 ml, 69.35 mmol).The solution was deoxygenated with argon and heated at 75° C. for fourhours. The solvent was then evaporated and the residue was purified on asilica gel column using 30% EtOAc/petroleum ether to give pure 7 (5.44g, 80% yield) as a white foam.

NMR: (CDCl₃) δ=2.16 (m, 1H), 2.84 (m, 1H), 3.8 Cm, 1H), 4.07 (m, 1H),4.60 (m, 1H), 6.19 (ddd, 1H), 7.92 (m, 1H).

2′-deoxy-α-L-5-fluorouridine (8)

A solution of compound 7 (5.44 g, 11.13 mmol) and NH₄F (4.12 g, 111.3mmol) in MeOH was stirred in an oil bath at 60° C. for 3 hours. Silicagel (3 g) was added and the mixture was evaporated to a dry powder. Thispowder was added to a silica column and eluted with 10-15% MeOH/CHCl₃ toproduce pure 8 (2.4 g, 87.6% yield) as a white foam.

NMR: (DMSO-d₆) δ=1.90 (m, 1H), 2.55 (m, 1H), 3.33 (m,2H), 4.19 (m, 2H),4.86(brs, 1H), 5.43(brs, 1H),6.10(dd, 1H), 8.15(d, 1H), 11.78(brs, 1H).

Example 2 Synthesis of 2′-deoxy-α-L-uridine 1-(2′, 3′,5′-tri-O-benzovl-α-L-arabinofuranosyl) uracil (9)

To a mixture of uracil (1.17 g, 10.49 mmol) and compound 2 (5 g) inanhydrous MeCN (100 ml) were successively added HMDS (1.77 ml, 8.39mmol), ClSiMe₃ (1.06 ml, 8.39 mmol), and SnCl₄ (1.47 ml, 12.58 mmol).The resulting clear solution was refluxed for one hour. Then the solventwas evaporated and the residue was dissolved in EtOAc (200 ml), washedwith H₂O, and saturated NaHCO₃ solution. The EtOAc layer was dried oversodium sulfate, filtered and evaporated to give the crude product, whichwas purified on a silica gel column using 40-50% EtOAc/petroleum etherto give pure 9 (3.66 g, 62.7% yield) as a white foam.

NMR: (CDCl₃) δ=4.70 (m, 1H), 5.77 (5, 1H), 5.80 (dd, 1H), 5.94 (t, 1H),6.20 (d, 1H), 7.40-8.10 (m, 16H), 8.58 (br s, 1H).

1-α-L-arabinofuranosyl-uracil (10)

To a solution of compound 8 (17.83 g, 32.03 mmol) in MeOH (400 ml),NaOMe (5.0 ml of a methanolic 25% w/v solution) was added and thesolution was stirred until the reaction was complete. The solvent wasthen evaporated and the residue was dissolved in H₂O (250 ml), washedwith ether and neutralized with Dowex 50 ion exchange resin. Afterfiltration of the resin, the aqueous solution was evaporated to givecompound 10 (7.4 g, 94.6% yield) as a white foam. This was used in thenext step without further purification.

1-[3′,5′-o-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-α-L-arabinofuranosyl]-uracil(11)

To a stirred suspension of 10 (7.4 g, 30.3 mmol) in pyridine was added1.3-dichloro-1,1,3,3-tetraisopropyldisiloxane (12.74 ml, 36.36 mmol).This was stirred at room temperature until the reaction was complete (5hours). The solvent was evaporated to a residue which was dissolved inEtOAC (500 ml) and washed successively with H₂O, 5% HCl, H₂O, saturatedNaHCO₃, and brine. After drying the EtOAc portion over Na₂SO₄, thesolution was filtered and evaporated to give the crude product 11 whichwas used in the next step without further purification.

1-[2′-o-phenoxythiocarbonyl-3′,5′-o-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-α-L-arabinofuranosyl]-uracil(12)

To a solution of 11 (30.3 mmol) in anhydrous MeCN were added4-dimethylaminopyridine (DMAP) (7.2 g, 58.78 mmol), andphenylchlorothionoformate (4.7 ml, 33.33 mmol). The solution was stirredat room temperature for 24 hours. Then, the solvent was evaporated to aresidue which was dissolved in EtOAc (750 ml) and washed successivelywith H₂O, 5% HCl, H₂O, saturated NaHCO₃, and brine. After drying theEtOAc portion over Na₂SO₄, the solution was filtered and evaporated toan oil. The oil was purified on a silica gel column using 30%EtOAc/petroleum ether to produce pure 12 (13.14 g, 74.5% yield) as awhite foam.

NMR: (CDCl₃) δ=4.04 (m, 2H), 4.38 (m, 1H), 4.73 (dd, 1H), 5.79 (dd, 1H),5.93 (d, 1H), 6.31 (dd, 1H), 7.08-7.33 (m, 6H), 9.2 (br s, 1H).

3′,5′-o-(1,1,3,3-tetraisopropyldisiloxane-1.3-diyl)-α-L-2′-deoxyuridine(13)

To a mixture of 12 (13.14 g, 21.09 mmol), in dry toluene (300 ml) wasadded AIBN (0.69 g, 4.2 mmol) followed by Bu₃SnH (28.4 ml, 105.4 mmol).The solution was deoxygenated with argon and heated at 75° C. for fourhours. The solvent was then evaporated and the residue was purified on asilica gel column using 30% EtOAc/petroleum ether to give pure 13 (9.29g, 88.4% yield) as a white foam.

NMR: (CDCl₃) δ=2.15 (2 t, 1H), 2.81 (m, 1H), 3.82 (dd, 1H), 4.05 (m,2H), 4.56 (q, 1H), 5.75 (dd, 1H), 6.16 (t, 1H), 7.69 (d, 1H), 9.38 (brs, 1H).

2′-deoxy-α-L-uridine (14)

A mixture of compound 13(9.2 g, 18.63 mmol) and NH₄F (6.9 g, 186.3 mmol)in MeOH (200 ml) was stirred in an oil bath at 60° C. for 3 hours.Silica gel (5 g.) was added and the mixture was evaporated to a drypowder. This powder was added to a silica column and eluted with 10-15%MeOH/CHCl₃ to produce pure 14 (3.70 g, 83% yield) as a white foam.

NMR: (DMSO-d₆) δ=1.87 (m, 1H), 2.56 (m, 1H), 3.41 (m, 2H), 4.15 (m, 1H),4.22 (m, 1H), 4.44 (t, 1H), 4.92 (t, 1H), 5.38 (d, 1H), 5.62 (d, 1H),6.09 (dd, 1H).

EXAMPLE 3 Synthesis of 2′-deoxy-α-L-cytidine

3′,5′-di-O-benzoyl-2′-deoxy-α-L-uridine (15)

A solution of BzCN (0.61 g, 4.67 mmol) in MeCN (10 ml) was addeddropwise to a suspension of compound 14 (0.43 g, 1.87 mmol) in MeCN (10ml) followed by Et₃N (0.1 ml). The reaction was stirred at roomtemperature for three hours after which time the solvent was evaporatedto dryness. The crude material was purified on a silica gel column using50% EtOAc/petroleum ether to give pure 15 (0.57 g, 70% yield) as yellowfoam.

NMR: (CDCl₃) δ=2.55 (d, 1H), 2.96 (dt, 1H), 4.56 (m, 2H), 4.86 (t, 1H),5.61 (d, 1H), 5.73 (dd, 1H), 6.31 (dd, 1H), 7.40-7.63 (m, 7H), 7.87-8.06(m, 4H), 8.82 (br s, 1H).

3′,5′-di-O-benzoyl-2′-deoxy-4-thio-α-L-uridine (16)

A boiling solution of compound 15 (0.54 g, 1.25 mmol) in anhydrousdioxane was treated with P₂S₅ (0.61 g, 2.75 mmol) and the mixture wasrefluxed under a nitrogen atmosphere for one hour. Remaining solids werefiltered from the hot solution and washed on the filter with additionaldioxane. The filtrate was evaporated to dryness and the crude productwas purified on a silica gel column using 30% EtOAc/petroleum ether togive pure 16 (0.42 9, 74% yield) as a yellow oil.

NMR: (CDCl₃) δ=2.59 (d, 1H), 2.93 (dt, 1H), 4.58 (m, 2H), 4.89 (t, 1H),5.63 is (d, 1H), 6.26 (dd, 1H), 6.41 (dd, 1H), 7.40-8.10 (m, 11H), 9.54(brs, 1H).

2′-deoxy-α-L-cytidine (17)

Compound 16 (0.42 g, 9.28 mmol) was treated with NH₃/MeOH (50 ml) in asteel bomb at 100° C. for 10 hours. After cooling, the solvent wasevaporated to dryness, the residue was dissolved in water (50 ml) andwashed with ether (3×50 ml). The water layer was treated with charcoal,filtered through Celite and evaporated to dryness by coevaporation withEtOH. The semi-solid obtained was crystallized from EtOH/ether to givecompound 17 (0.18 g, 85.7% yield).

NMR: (DMSO-d₆) δ=1.86 (Cd,H), 2.50 (m, 1H), 3.40 (m, 1H), 4.12 (m, 1H),4.15 (m, 1H), 4.86 (t, 1H), 5.21 (d, 1H), 5.69 (d, 1H), 6.03 (dd, 1H),7.02 (br d, 1H), 7.74 (d, 1H).

N⁴-benzoyl-2′-deoxy-α-L-cytidine (18)

ClSiMe₃ (2.3 ml, 18.05 mmol) was added dropwise over 30 minutes to astirring suspension of compound 17 (0.82 g, 3.61 mmol) in pyridine (50ml) chilled in an ice bath. BzCl (2.1 ml, 18.05 mmol) was then addeddropwise and the reaction mixture was cooled at room temperature for twohours. The reaction mixture was again cooled in an ice bath and coldwater (10 ml) was added dropwise. Fifteen minutes later, concentratedNH₄0H (10 ml) was added to produce a solution of ammonia of aconcentration of about 2M. Thirty minutes after the addition of theammonia solution, a solvent was evaporated, dissolved in water andwashed with ether. Evaporation of this aqueous solution provided thecrude product (18) which was used in the next step without furtherpurification.

EXAMPLE 4 Synthesis of Dimers

The dimers were prepared from the monomeric materials by the generalscheme shown in Scheme 2.

A. α-L, β-D 5 FUdR Dimer

5′-O-dimethoxvtrityl-α-L-5-fluoro-2′-deoxyuridine (20a)

α-L-5-fluoro-2′-deoxyuridine (8) (500 mg, 2.0 mmol) was dissolved in 10ml of dry, distilled pyridine. To this solution was added4,4′-dimethoxytrityl chloride (813 mg, 2.4 mmol) and4-dimethylaminopyridine (DMAP) (50 mg, 0.4 mmol). The mixture wasstirred under an argon atmosphere for 16 hours. After this time, thepyridine was stripped off in vacua. The residue was dissolved in EtOAc(50 ml). The organic layer was washed with saturated NaHCO₃, water andwith brine. The organic layer was dried over Na₂SO₄, filtered andevaporated in vacuo to a residue which was purified on a silica gelcolumn using 10% MeOH/CHCl₃. Pure fractions were pooled and evaporatedto give the pure product as an off-white foam (679 mg, 86% yield).Rf=0.48 in 10% MeOH/CHCl₃.

NMR: (DMSO-d₆) δ=2.3 (dd, 1H), 2.72-2.81 (m, 1H), 3.15-3.26 (m, 2H),3.75 (s, 6H), 4.45 (m, 2H), 6.23 (dd, 1H), 6.92 (d, 1H), 7.2-7.3 (m, 13H), 7.94 (d, 1H).

5′-O-dimethoxvtrityl-α-L-5-fluoro-2′-deoxyuridine-3′-N,N-diisopropylmethoxy phosphoramidite (21a)

The 5′-O-dimethoxytrityl-α-L-5-fluoro-2′-deoxyuridine (20a, 548 mg, 1mmol) was dissolved in anhydrous dichloromethane (20 ml).N,N-diisopropylethylamine (700 pi, 4 mmol) was added through a septum,followed by chloro-N,N-diisopropylmethoxyphosphine (290 pi, 1.5 mmol),10 under an argon atmosphere. The reaction was stirred for 30 minutes.The solvent was evaporated and the residue was partitioned between an80% EtOAc/triethylamine mixture and brine. The organic layer was washedwith saturated NaHCO₃ solution and brine. The organic residue wasevaporated to dryness and the residue was purified on a silica gelcolumn using a mixture of dichloromethane, EtOAc and triethylamine(45:45:10;Rf=0.69). The product (390 mg) was isolated as a yellow foamand it was used in the next step without further purification.

3′-Acetoxy-β-D-5-fluoro-2′-deoxyuridine (24a)

β-D-5-fluoro-2′-deoxyuridine (500 mg, 2.2 mmol) was dissolved in 10 20ml of dry, distilled pyridine. To this solution was added4,4′-dimethoxytrityl chloride (813 mg, 2.4 mmole) and4-dimethylaminopyridine (DMAP) (50 mg, 0.4 mmole). The mixture wasstirred at room temperature for 16 hours. The pyridine was stripped offin vacuo. The residue was dissolved in dichloromethane (50 ml). Theorganic layer was washed with 0.3 NHCl, brine, saturated NaHCO₃, andagain with brine. The organic layer was dried over Na₂SO₄, filtered andevaporated in vacuo to a residue which was purified on a silica gelcolumn, eluting with 10% MeOH/CHCl₃. Pure fractions were pooled andevaporated to give the pure product as an off-white foam (685 mg, 86%yield). This material was dissolved in pyridine (12 ml) and treated withacetic anhydride (2.5 ml) for 3 hours at room temperature. The solventwas evaporated, and the residue was dissolved in ethyl acetate. Theethyl acetate was washed as described above, dried over sodium sulfateand evaporated. The residue was then treated with 80% acetic acid (10ml) for 2.5 hours at room temperature. The solvent was evaporated invacuo and the residue was chromatographed on silica gel, eluting with10% MeOH/CHCl₃ to give pure 24a as a white foam, yield 422 mg.

NMR: (DMSO-d₆) δ=1.95 (s, 3H), 2.08-2.24 (m, 2H), 3.65-3.9 (m, 2H), 4.4510 (m, 1H), 4.72 (m, 1H), 6.24 (dd, 1H), 8.24 (d, 1H).

5′-O-dimethoxytrityl-3′-[O-(3′-O-acetyl)-β-D-5-fluoro-2′-deoxyuridinyl]-α-L-5-fluoro-2′-deoxyuridine(25a)

The 3′-O-acetyl-β-D-5-fluoro-2′-deoxyuridine (188 mg, 0.65 mmol) wasdissolved in dry acetonitrile (5 ml). Sublimed 1H-tetrazole (80 mg) wasadded and the mixture was stirred under an argon atmosphere for 15minutes. The solution of 21a (380 mg, 0.54 mmol), dissolved in 5 ml ofdry acetonitrile was added via syringe to the reaction solution over 5minutes.

The mixture was allowed to stir at room temperature for three hours. Theacetonitrile was evaporated in vacuo to a residue. This residue wastriturated with a 70% EtOAc/ether mixture. The undissolved tetrazole wasfiltered off and the filtrate was evaporated to give a dry yellow foam(468 mg). This foam was used in the next step without furtherpurification.

(3′-acetoxy-β-D-5-fluoro-2′-deoxyuridinyl)-α-L-5-fluoro-2′-deoxyuridinemethyl phosphonate ester (26a)

The dimer, 25a (504 mg), was dissolved in 8 ml of THF and 2 ml ofpyridine containing 0.2 ml of water. Iodine crystals (26 mg) were addedand the contents of the loosely stoppered flask were allowed to stir for2.1 hours. Excess iodine was discharged by the addition of a few dropsof saturated sodium thiosulfate. The reaction mixture was thenevaporated to dryness.

The crude product was dissolved in EtOAc washed with saturated NaHCO₃solution and brine. The organic layer was dried over Na₂SO₄, filteredand evaporated in vacuo. The residue (530 mg) was dissolved in 10 ml of80% acetic acid/water solution and was stirred until the reaction wascompleted. The solvent was evaporated and the residue was purified on asilica gel column, using 20% MeOH/CHCl₃. Fractions containing one spotby TLC (10% MeOH/CHCl₃ Rf=0.35) were pooled and evaporated to give thepure product (316 mg).

NMR: (CD₃OD) δ=2.08 (s, 3H), 2.25-2.45 (m, 3H), 2.65-2.72 (m, 1H), 3.60(m, 2H), 3.80 (2d, 3H), 4.18 (m, 1H), 4.28 (m, 1H), 4.35 (dd, 1H), 4.62(dd, 1H), 5.05 (dd, 1H), 5.23 (m, 1H), 6.13 (m, 1H), 6.18 (m, 1H), 7.85(m, 2H).

P³¹NMR: (CD₃OD) δ=0.77 (s), 1.16 (s).

3′-O-(β-D-5-fluoro-2′-deoxyuridinyl)-α-L-5-fluoro-2′-deoxyuridine (27a)

The O-protected dimer, 26a (280 mg) was treated with 20 ml of saturatedmethanolic ammonia at room temperature until the reaction was completedat room temperature. The solvent was stripped off in vacuo and theresidue was purified on DEAE cellulose ion exchange column usinggradient of NH₄CO₃ buffer from 0.02-0.2M. Pure fractions were evaporatedat 40° C. in high vacuo to dryness to give the pure product (162 mg).

NMR: (D₂O) δ=2.2-2.4 (m, 3H), 2.65-2.71 (m, 1H), 3.65 (m, 2H), 4.01 (m,1H), 4.11 (t, 1H), 4.45 (m, 1H), 4.65 (t, 1H), 6.14 (d, 1H), 6.24 (td,1H), 8.06 (d, 1H), 8.02 (d, 1H).

³¹P NMR: (D₂O) δ=0.04 (s).

B. β-D, α-L 5FUdR Dimer

5′-O-dimethoxytrityl-3′-[O-(3′-O-acetyl)-α-L-5-fluoro-2′-deoxguridinyl]-β-D-5-fluoro-2′-deoxyuridine(25b)

The 3′-O-acetyl-α-L-5-fluoro-2′-deoxyuridine, 24b (188 mg, 0.65 mmol)was dissolved in dry acetonitrile (5 ml). Sublimed 1H-tetrazole (80 mg)was added and the mixture was stirred under an argon atmosphere for 15minutes. The solution of 21b (380 mg, 0.54 mmol), dissolved in 5 ml ofdry acetonitrile was added via syringe to the reaction solution over 5minutes. The mixture was allowed to stir at room temperature for threehours. The acetonitrile was evaporated in vacuo to a residue. Thisresidue was triturated with a 70% EtOAc/ether mixture. The undissolvedtetrazole was filtered off and the filtrate was evaporated to give a dryyellow foam (484 mg). This foam was used in the next step withoutfurther purification.

(3′-acetoxy-α-L-5-fluoro-2′-deoxyuridinyl)-β-D-5-fluoro-2′-deoxyuridinemethyl phosphate ester (26b)

The dimer, 25b (526 mg), was dissolved in 8 ml of THF and 2 ml ofpyridine containing 0.2 ml of water. Iodine crystals (26 mg) were addedand the contents of the loosely stoppered flask were allowed to stir for1 hour. Excess iodine was discharged by the addition of a few drops ofsaturated sodium thiosulfate. The reaction mixture was then evaporatedto dryness. The crude product was dissolved in EtOAc washed withsaturated NaHCO₃ solution and brine. The organic layer was dried overNa₂SO₄, filtered and evaporated in vacuo. The residue (578 mg) wasdissolved in 10 ml of 80% acetic acid/water solution and was stirred forthree hours. The solvent was evaporated and the residue was purified ona silica gel column, using 10-15% MeOH/CHCl₃. Fractions containing onespot by TLC (10% MeOH/CHCl₃ Rf=0.35) were pooled and evaporated to givethe pure product (342 mg).

NMR: (DMSO-d₆) δ=1.98 (s, 3H), 2.2-2.4 (m, 3H), 2.62-2.71 (m, 1H),3.5-3.8(m,4H), 4.02(m, 1H),4.42(m,2H),6.10(dd, 1H), 6.26(dt, 1H), 8.00(d, 1H), 8.04 (d, 1H).

3′-O-(α-L-5-fluoro-2′-deoxyuridinyl)-β-D-5-fluoro-2′-deoxyuridine (27b)

The O-protected dimer, 26b (170 mg) was treated with 20 ml of saturatedmethanolic ammonia at room temperature until the reaction was completed.The solvent was stripped off in vacuo and the residue was purified onDEAE cellulose ion exchange column using gradient of NH₄CO₃ buffer from0.02-0.2M. Pure fractions were evaporated at 40° C. in high vacuo todryness to give the pure product (89 mg).

NMR: (D₂O) δ=2.2-2.4 (m, 3H), 2.65-2.71 (m, 1H), 3.54-3.85 (m, 5H), 4.05(t, 1H), 4.42 (m, 2H), 6.06 (dd, 1H), 6.23 (dt, 1H), 8.00 (d, 1H), 8.04(d, 1H).

C. α-L uridine, β-D 5 FUdR dimer5′-O-(di-p-methoxytrityl)-2′-deoxy-α-L-uridine (20c)

α-L-2′-deoxyuridine (1.5 g, 6.57 mmol) was dissolved in 25 ml of dry,distilled pyridine. To this solution was added 4,4′-dimethoxytritylchloride (2.9 g, 7.89 mmol) and 4-dimethylamino pyridine (DMAP) (160 mg,1.31 mmol). The mixture was stirred under an argon atmosphere for 16hours. After this time, the pyridine was stripped off in vacuo. Theresidue was dissolved in EtOAc (150 ml). The organic layer was washedwith saturated NaHCO₃, water and again with brine. The organic layer wasdried over Na₂SO₄, filtered and evaporated in vacuo to a residue whichwas purified on a silica gel column using 5% MeOH/CHCl₃. Pure fractionswere pooled and evaporated to give the pure product as an off-white foam(2.84 g, 81% yield).

NMR: (CDCl₃-d₆) δ=2.29 (d, 1H), 2.70 (m, 2H), 3.17 (m, 2H), 3.78 (s,6H), 4.44 (m, 2H), 5.63 (d, 1H), 6.19 (d, 1H), 6.83 (d, 4H), 7.28 (m, 9H), 7.68 (d, 1H), 9.30 (br s, 1H).

5′-O-(dimethoxytrityl)-α-L-2′-deoxyuridine-3′-N,N-diisopropylmethoxyphosphoramidite (21c).

The 5′-O-dimethoxytrityl-α-L-2′-deoxyuridine (2.35 g, 4.43 mmol) wasdissolved in anhydrous dichloromethane (50 ml).N,N-diisopropylethylamine (3.1 ml, 17.72 mmol) was added through aseptum, followed by chloro-N,N-diisopropylmethoxyphosphine (1.3 ml, 6.64mmol), under an argon atmosphere. The reaction was stirred for 30minutes. The solvent was evaporated and the residue was partitionedbetween an 80% EtOAc/triethylamine mixture and brine. The organic layerwas washed with saturated NaHCO₃ solution and brine. The organic residuewas evaporated to dryness and the residue was purified on a silica gelcolumn using a 5 mixture of dichloromethane, EtOAc and triethylamine(40:50:10; Rf=0.69). The product was isolated quantitatively as a yellowfoam and it was used in the next step without further purification.

5′-O-dimethoxytrityl-3′-[O-(3′-O-acetyl)-β-D-5-fluoro-2′-deoxyuridinyl]-2′-deoxy-α-L-uridine(25c)

The 3′-O-acetyl-β-D-5-fluoro-2′-deoxyuridine (0.95 g, 3.29 mmol) wasdissolved in dry acetonitrile (125 ml). Sublimed 1H-tetrazole (350 mg,4.91) was added and the mixture was stirred under an argon atmospherefor 15 minutes. The solution of 21c (4.91 mmol), dissolved in 5 ml ofdry acetonitrile was added via syringe to the reaction solution over 5minutes. The mixture was allowed to stir at room temperature for threehours. The acetonitrile was evaporated in vacuo to a residue. Thisresidue was triturated with a 70% EtOAc/ether mixture. The undissolvedtetrazole was filtered off and the filtrate was evaporated to give a dryyellow foam. This compound was further purified on a silica gel columnusing 5% MeOH/CHCl₃ to give the pure product (2.81 g, 97% yield).

3′-acetoxy-β-D-5′-fluoro-2′-deoxyuridinyl)-α-L-2′-deoxyuridine methylphosphate ester (26c)

The dimer, 25c (2.81 g, 3.2 mmol), was dissolved in a mixture ofTHF:pyridine:water (25:6:0.6). Iodine crystals (150 mg) were added andthe contents of the loosely stoppered flask were allowed to stir for 1hour. Excess iodine was discharged by the addition of a few drops ofsaturated sodium thiosulfate. The reaction mixture was then evaporatedto dryness. The crude product was dissolved in EtOAc washed withsaturated NaHCO₃ solution and brine. The organic layer was dried overNa₂SO₄, filtered and evaporated in vacuo. The residue (1.48 g) wasdissolved in 25 ml of 80% acetic acid/water solution and was stirreduntil the reaction was completed. The solvent was evaporated and theresidue was purified on a silica gel column, using 10% MeOH/CHCl₃.Fractions containing one spot by TLC (10% MeOH/CHCl₃ Rf=0.4) were pooledand evaporated to give the pure product (0.465 g, 25% yield).

NMR: (CD₃OD) δ=2.09 (d, 3H), 2.40 (m, 3H), 2.80 (m, 1H), 3.78 (dd, 3H),4.30 (m, 3H), 4.63 (m, 1H), 5.05 (m, 1H), 5.23 (m, 1H), 5.70 (d, 1H),6.13 (m, 1H), 6.20 (m, 1H), 7.73 (d, 1H), 7.82 (d, 1H).

P³¹NMR: (CD₃OD) δ=0.56 (s), 0.84 (s).

3′-O-(β-D-5-fluoro-2′-deoxyuridinyl)-α-L-2′-deoxyuridine (27c)

The O-protected dimer, 26c (465 mg, 0.78 mmol) was treated with 50 ml ofsaturated methanolic ammonia at room temperature until the reaction wascompleted. The solvent was stripped off in vacuo and the residue waspurified on DEAE cellulose ion exchange column using gradient of NH₄CO₃buffer from 0.02-0.2M. Pure fractions were evaporated at 40° C. in highvacuo to dryness to give the pure product (370 mg, 87.7% yield).

N NMR: (CD₃OD) δ=2.23 (m, 2H), 2.29 (d, 1H), 2.73(m, 1H), 4.0 (d, 2H),4.42 (m, 1H), 4.56 (m, 1H), 4.81 (m, 1H), 5.69 (d, 1H), 6.24 (m, 2H),7.85 (d, 1H), 8.02 (d, 1H).

P³¹ NMR: (CD₃OD) δ=1.25 (s).

D. β-L. β-L 5 FUdR dimer

5′-O-dimethoxytrityl-β-L-5-fluoro-2′-deoxyuridine (20d)

β-L-5-fluoro-2′-deoxyuridine (19d, 1.42 g, 5.77 mmol) was dissolved in25 ml of dry, distilled pyridine. To this solution was added4,4′-dimethoxytrityl chloride (2.34 g, 6.92 mmol) and 4-dimethylaminopyridine (DMAP) (140 mg, 1.15 mmol). The mixture was stirred under anargon atmosphere for 16 hours. After this time, the pyridine wasstripped off in vacuo. The residue was dissolved in EtOAc (100 ml). Theorganic layer was washed with saturated NaHCO₃, and with brine. Theorganic layer was dried over Na₂SO₄, filtered and evaporated in vacuo toa residue which was purified on a silica gel column using 5% MeOH/CHCl₃.Pure fractions were pooled and evaporated to give the pure product as anoff-white foam (2.88 g, 88.7% yield).

NMR: (CDCl₃) δ=2.25 (m, 1H), 2.50 (m, 1H), 3.50 (m, 2H), 3.80 (s, 6H),4.08 (m, 1H), 4.58 (m, 1H), 6.30 (t, 1H), 6.84 (d, 4H), 7.28 (m, 9 H),7.82 (d, 1H), 8.58 (br s, 1H).

5′-O-dimethoxytrityl-β-L-5-fluoro-2′-deoxyuridine-3′-N,N-diisopropylmethoxyphosphoramidite (21d)

The 5′-O-dimethoxytrityl-β-L-5-fluoro-2′-deoxyuridine (20d, 840 mg, 1.53mmol) was dissolved in anhydrous dichloromethane (50 ml).N,N-diisopropylethylamine (1.1 ml, 6.13 mmol) was added through aseptum, followed by chloro-N,N-diisopropylmethoxyphosphine (0.42 ml, 2.3mmol), under an argon atmosphere. The reaction was stirred for 30minutes. The solvent was evaporated and the residue was partitionedbetween an 80% EtOAc/triethylamine mixture and brine. The organic layerwas washed with saturated NaHCO₃ solution and brine. The organic residuewas evaporated to dryness and the residue was purified on a silica gelcolumn using a mixture of dichloromethane, EtOAc and triethylamine(45:45:10; Rf=0.69). The product (700 mg, 65%) was isolated as a yellowfoam and it was used in the next step without further purification.

5′-O-dimethoxytrityl-3′-[O-(3′-O-acetyl)-β-L-5-fluoro-2′-deoxyuridinyl]-β-L-5-fluoro-2′-deoxyuridine(25d)

The 3′-acetyl-β-L-5-deoxyuridine, 24d (330 mg, 1.15 mmol) was dissolvedin dry acetonitrile (50 ml). Sublimed 1H-tetrazole (120 mg, 1.77 mmol)was added and the mixture was stirred under an argon atmosphere for 15minutes. The solution of 21d (950 mg, 1.36 mmol), dissolved in 5 ml ofdry acetonitrile was added via syringe to the reaction solution over 5minutes. The mixture was allowed to stir at room temperature for threehours. The acetonitrile was evaporated in vacuo to a residue. Thisresidue was triturated with a 70% EtOAc/ether mixture. The undissolvedtetrazole was filtered off and the filtrate was evaporated to give a dryyellow foam.

This foam was purified on a silica gel column using 5% MeOH/CHCl₃ togive the pure product (960 mg, 93% yield).

NMR: (CDCl₃) δ=2.10 (d, 3H), 2.28 (m, 2H), 2.49 (m, 2H), 3.42 (m, 3H),3.51 (dd, 3H), 3.76 (s, 6H), 4.07 (m, 1H), 4.55 (m, 1H), 4.87 (m, 1H),5.23 (m, 1H), 6.30 (m, 2H), 6.84 (d, 4H), 7.30 (m, 9H), 7.82 (m, 2H).

(3′-acetoxy-β-L-5-fluoro-2′-deoxyuridinyl)-β-L-5-fluoro-2′-deoxyuridinemethyl phosphate ester (26d)

The dimer, 25d (960 mg, 1.07 mmol), was dissolved in a mixturecontaining THF:pyridine:water (12:3:0.3). Iodine crystals (50 mg) wereadded and the contents of the loosely stoppered flask were allowed tostir for 1 hour. Excess iodine was discharged by the addition of a fewdrops of saturated sodium thiosulfate. The reaction mixture was thenevaporated to dryness. The crude product was dissolved in EtOAc washedwith saturated NaHCO₃ solution and brine. The organic layer was driedover Na₂SO₄, filtered and evaporated in vacuo. The residue (530 mg) wasdissolved in 20 ml of 80% acetic acid/water solution and was stirreduntil the reaction was completed. The solvent was evaporated and theresidue was purified on a silica gel column, using 10% MeOH/CHCl₃.Fractions containing one spot by TLC (10% MeOH/CHCl₃ Rf=0.35) werepooled and evaporated to give the pure product (310 mg, 46% yield).

NMR: (CD₃OD) δ=2.08 (s, 3H), 2.35-2.54 (m, 4H), 3.79 (m, 2H), 3.83 (dd,3H), 4.18 (m, 2H), 5.08 (m, 1H), 5.29 (m, 1H), 6.24 (m, 1H), 7.86 (dd,1H), 8.19 (dd, 1H).

P³¹ NMR: (CD₃OD) δ=0.82 (s), 1.03 (s).

3′-O-(β-L-5-fluoro-2′-deoxyuridinyl)-β-L-5-fluoro-2′-deoxyuridine (27d)

The O-protected dimer, 26d (300 mg, 0.49 mmol) was treated with 50 ml ofsaturated methanolic ammonia at room temperature until the reaction wascompleted. The solvent was stripped off in vacuo and the residue waspurified on DEAE cellulose ion exchange column using gradient of NH₄CO₃buffer from 0.02-0.2M. Pure fractions were evaporated at 40° C. in highvacuo to dryness to give the pure product (240 mg, 85% yield). NMR:(CD₃OD) δ=2.25 (m, 3H), 2.50 (m, 1H), 3.79 (d, 2H), 4.03 (m, 1H), 4.08(m, 2H), 4.18 (m, 1H), 4.44 (m, 1H), 4.90 (m, 1H), 6.25 (t, 1H), 8.01(d, 1H), 8.24 (d, 1H).

P³¹ NMR: (CD₃OD) δ=0.18 (s).

E. β-L, β-D 5 FUdR dimer

5′-O-dimethoxytrityl-3′-[O-(3′-O-acetyl)-β-D-5-fluoro-2′-deoxyuridinyl]-(-L-5-fluoro-2′-deoxyuridine(25e)

The 3′-O-acetyl-⊖-D-5-fluoro-2′-deoxyuridine (250 mg, 0.97 mmol) wasdissolved in dry acetonitrile (50 ml). Sublimed 1H-tetrazole (100 mg,1.46 mmol) was added and the mixture was stirred under an argonatmosphere for 15 minutes. The solution of 21e (1.02 g, 1.46 mmol),dissolved in 5 ml of dry acetonitrile was added via syringe to thereaction solution over 5 minutes. The mixture was allowed to stir atroom temperature for three hours. The acetonitrile was evaporated invacuo to a residue. This residue was triturated with a 70% EtOAc/ethermixture. The undissolved tetrazole was filtered off and the filtrate wasevaporated to give a dry yellow foam. This foam was purified on a silicagel column using 5% MeOH/CHCl₃ to give the pure product quantitatively.

(3′-acetoxy-β-D-5-fluoro-2′-deoxyuridinyl)-β-L-5-fluoro-2′-deoxyuridinemethyl phosphonate ester (26e)

The dimer in reduced form, 25 (700 mg, 0.78 mmol), was dissolved in amixture containing THF:pyridine:water (25:6:0.6). Iodine crystals (100mg) were added and the contents of the loosely stoppered flask wereallowed to stir for 2.5 hours. Excess iodine was discharged by theaddition of a few drops of saturated sodium thiosulfate. The reactionmixture was then evaporated to dryness. The crude product was dissolvedin EtOAc washed with saturated NaHCO₃ solution and brine. The organiclayer was dried over Na₂SO₄, filtered and evaporated in vacuo. Theresidue was dissolved in 25 ml of 80% acetic acid/water solution and wasstirred until the reaction was completed. The solvent was evaporated andthe residue was purified on a silica gel column, using 10% MeOH/CHCl₃.Fractions containing one spot by TLC (10% MeOH/CHCl₃ Rf=0.35) werepooled and evaporated to give the pure product (340 mg, 71.4% yield).

NMR: (DMSO-d₆) δ=2.06 (s, 3H), 2.37 (m, 4H), 3.45 (m, 2H), 3.65 (d, 3H),4.20 (m, 3H), 4.95 (m, 1H), 5.30 (m, 1H), 5.96 (m, 1H), 6.15 (t, 2H),7.99 (d, 1H), 8.16 (d, 1H), 1 1.90(br s, 2H).

P³¹ NMR: (DMSO-d₆) δ=1.93 (s), 2.01 (s).

3′-O-(β-D-5-fluoro-2′-deoxyuridinyl)-3-L-5-fluoro-2′-deoxyuridine (27e)

The O-protected dimer, 26e (340 mg, 0.57 mmol) was treated with 100 mlof saturated methanolic ammonia at room temperature until the reactionwas completed. The solvent was stripped off in vacuo and the residue waspurified on DEAE cellulose ion exchange column using gradient of NH₄CO₃buffer from 0.02-0.2M. Pure fractions were evaporated at 40° C. in highvacuo to dryness to give the pure product (200 mg, 66.9% yield).

NMR: (CD₃OD) δ=2.20 (m, 3H), 2.53 (m, 1H), 3.79 (d, 2H), 4.05 (m, 3H),4.16 (m, 1H), 4.45 (m, 1H), 6.27 (t, 2H), 8.01 (d, 1H), 8.04 (d, 1H),8.26 (d, 1H).

5′-O-(dimethoxytrityl)-α-L-5-fluoro-2′-deoxyuridine-3′-NN-diisopropylcyanoethyl phosphoramidite (21f)

The 5′-o-dimethoxytrityl-α-L-5-fluoro-2′-deoxyuridine (1.48 g, 2.71mmol) was dissolved in anhydrous dichloromethane (50 ml).N,N-diisopropylethylamine (1.9 ml, 10.84 mmol) was added through aseptum, followed by 2′-cyanoethyl-N,N-diisopropylchlorophosphoramidite(0.78 ml, 3.52 mmol), under an argon atmosphere. The reaction wasstirred for 30 minutes. The solvent was evaporated and the residue waspartitioned between an 80% EtOAc/triethylamine mixture and brine. Theorganic layer was washed with saturated NaHCO₃ solution and brine. Theorganic residue was evaporated to dryness and the residue was purifiedon a silica gel column using a mixture of dichloromethane, EtOAc andtriethylamine (45:45:10: Rf=0.7). The product was isolatedquantitatively as a yellow foam and it was used in the next step withoutfurther purification.

5′-O-dimethoxytrityl-3′-[O-(5′-O-dimethoxytrityl)-β-D-5-fluoro-2′-deoxyuridinyl]-α-L-5-fluoro-2′-deoxyuridine(25f)

The 5′-O-dimethoxytrityl-β-D-5-fluoro-2′-deoxyuridine (0.44 g, 0.81mmol) was dissolved in dry acetonitrile (20 ml). Sublimed 1H-tetrazole(90 mg) was added and the mixture was stirred under an argon atmospherefor 15 minutes. The solution of 21f (0.51 mg, 0.67 mmol), dissolved in10 ml of dry acetonitrile was added via syringe to the reaction solutionover 5 minutes. The mixture was allowed to stir at room temperature forthree hours. The acetonitrile was evaporated in vacuo to a residue. Thisresidue was triturated with a 70% EtOAc/ether mixture. The undissolvedtetrazole was filtered off and the filtrate was evaporated to give a dryyellow foam (970 mg). This foam was used in the next step withoutfurther purification.

(β-D-5-fluoro-2′-deoxyuridinyl)-α-L-5-fluoro-2′-deoxyuridine cyanoethylphosphonate ester (26f)

The dimer, 25f (970 mg), was dissolved in 16 ml of THF and 4 ml ofpyridine containing 0.4 ml of water. Iodine crystals (50 mg) were addedand the contents of the loosely stoppered flask were allowed to stir for1 hour.

Excess iodine was discharged by the addition of few drops of saturatedsodium thiosulfate. The reaction mixture was then evaporated to dryness.

The crude product was EtOAc washed with saturated NaHCO₃ solution andbrine. The organic layer was dried over Na₂SO₄, filtered and evaporatedin vacuo. The residue was dissolved in 20 ml of 80% acetic acid/watersolution and was stirred until the reaction was completed. The solventwas evaporated and the residue was purified on a silica gel column,using 10-15% MeOH/CHCl₃. Fractions containing one spot by TLC (10%MeOH/CHCl₃ Rf=0.35) were pooled and evaporated to give the pure product(330 mg).

3′-O-(13-D-5-fluoro-2′-deoxyuridinyl)-α-L-5-fluoro-2′-deoxyuridine (27f)

The O-protected dimer, 26f (200 mg) was treated with 20 ml ofconcentrated ammonia solution until the reaction is completed. Thesolvent was stripped off in vacuo and the residue was purified on DEAEcellulose ion exchange column using gradient of NH₄CO₃ buffer from0.02-0.2M. Pure fractions were evaporated at 40° C. in high vacuo todryness to give the pure product (79 mg).

NMR: (CD₃OD) δ=2.45 (m, 3H), 2.69 (m, 1H), 3.67 (m, 2H), 3.76 (m, 2H),4.13 (t, 1H), 4.65 (m, 2H), 6.19 (m, 2H), 7.98 (td, 2H).

P³¹ NMR: (D₂O) δ=−1.0(s)

5′-O-dimethoxytrityl-3′-[O-(3′-O-acetyl)-β-L-5-fluoro-2′-deoxyuridinyl]-α-L-5-fluoro-2′-deoxyuridine(25g)

The 3′-O-acetyl-β-D-5-fluoro-2′-deoxyuridine (0.19 g, 0.67 mmol) wasdissolved in dry acetonitrile (20 ml). Sublimed 1H-tetrazole (70 mg) wasadded and the mixture was stirred under an argon atmosphere for 15minutes. The solution of 21f (0.51 mg, 0.67 mmol), dissolved in 10 ml ofdry acetonitrile was added via syringe to the reaction solution over 5minutes. The mixture was allowed to stir at room temperature for threehours. The acetonitrile was evaporated in vacuo to a residue. Thisresidue was triturated with a 70% EtOAc/ether mixture. The undissolvedtetrazole was filtered off and the filtrate was evaporated to give a dryyellow foam (611 mg). This foam was used in the next step withoutfurther purification.

(3′-acetoxy-β-L-5-fluoro-2′-deoxyuridinyl)-α-L-5-fluoro-2′-deoxyuridinecyanoethyl phosphonate ester (26a)

The dimer, 25g (611 mg), was dissolved in 8 ml of THF and 2 ml ofpyridine containing 0.2 ml of water. Iodine crystals (30 mg) were addedand the contents of the loosely stoppered flask were allowed to stir for1 hour. Excess iodine was discharged by the addition of few drops ofsaturated sodium thiosulfate. The reaction mixture was then evaporatedto dryness. The crude product was EtOAc washed with saturated NaHCO₃solution and brine. The organic layer was dried over Na₂SO₄, filteredand evaporated in vacuo. The residue was dissolved in 20 ml of 80%acetic acid/water solution and was stirred until the reaction wascompleted. The solvent was evaporated and the residue was purified on asilica gel column, using 10-15% MeOH/CHCl₃. Fractions containing onespot by TLC (10% MeOH/CHCl₃ Rf=0.35) were pooled and evaporated to givethe pure product (200 mg).

3′-O-(β-L-5-fluoro-2′-deoxyuridinyl)-α-L-5-fluoro-2′-deoxyuridine (27g)[α-L β-L 5FUdR Dimer]

The o-protected dimer, 26g (200 mg) was treated with 20 ml ofconcentrated ammonia solution until the reaction is completed. Thesolvent was stripped off in vacuo and the residue was purified on DEAEcellulose ion exchange column using gradient of NH₄CO₃ buffer from0.02-0.2M. Pure fractions were evaporated at 40° C. in high vacuo todryness to give the pure product (134 mg).

NMR: (D₂O) δ=2.30 (m, 3H), 2.71 (m, 1H), 3.65 (m, 2H), 4.03 (m, 2H),4.08 (t, 1H), 4.47 (m, 1H), 4.68 (m, 2H), 6.13 (d, 1H), 6.24 (td, 1H),7.89 (d, 1H), 7.95 (d, 1H).

P³¹ NMR: (D₂O) δ=0.32(s)

5′-O-(dimethoxytrityl)-β-L-2′-deoxyuridine-3′-N,N-diisopropylmethoxyphosphoramidite (21h)

The 5′-O-dimethoxytrityl-α-L-2′-deoxyuridine (1.0 g, 1.88 mmol) wasdissolved in anhydrous dichloromethane (50 ml).N,N-diisopropylethylamine (1.31 ml, 7.55 mmol) was added through aseptum, followed by chloro-N,N-diisopropylmethoxyphosphine (0.55 ml,2.83 mmol), under an argon atmosphere. The reaction was stirred for 30minutes. The solvent was evaporated and the residue was partitionedbetween an 80% EtOAc/triethylamine mixture and brine. The organic layerwas washed with saturated NaHCO₃ solution and brine. The organic residuewas evaporated to dryness and the residue was purified on a silica gelcolumn using a mixture of dichloromethane, EtOAc and triethylamine(50:40:10; Rf=0.8). The product was isolated quantitatively as a yellowfoam and it was used in the next step without further purification.

5′-O-dimethoxytrityl-3′-[O-(3′-O-acetyl)-β-D-5-fluoro-2′-deoxyuridinyl]-β-L-2′-deoxyuridine(25h)

The 3′-O-acetyl-β-D-5-fluoro-2′-deoxyuridine (0.54 g, 1.88 mmol) wasdissolved in dry acetonitrile (50 ml). Sublimed 1H-tetrazole (200 mg)was added and the mixture was stirred under an argon atmosphere for 15minutes. The solution of 21h (1.88 mmol), dissolved in 15 ml of dryacetonitrile was added via syringe to the reaction solution over 5minutes. The mixture was allowed to stir at room temperature for threehours. The acetonitrile was evaporated in vacuo to a residue. Thisresidue was triturated with a 70% EtOAc/ether mixture. The undissolvedtetrazole was filtered off and the filtrate was evaporated to give a dryyellow foam (1.08 g). This foam was used in the next step withoutfurther purification.

(3′-acetoxy-β-D-5-fluoro-2′-deoxyuridinyl)-β-L-2′-deoxyuridine methylphosphonate ester (26h)

The dimer, 25h (1.08 g), was dissolved in 15 ml of THF and 3 ml ofpyridine containing 0.3 ml of water. Iodine crystals (100 mg) were addedand the contents of the loosely stoppered flask were allowed to stir for1 hour. Excess iodine was discharged by the addition of few drops ofsaturated sodium thiosulfate. The reaction mixture was then evaporatedto dryness. The crude product was EtOAc washed with saturated NaHCO₃solution and brine. The organic layer was dried over Na₂SO₄, filteredand evaporated in vacuo. The residue was dissolved in 25 ml of 80%acetic acid/water solution and was stirred until the reaction wascompleted. The solvent was evaporated and the residue was purified on asilica gel column, using 10-15% MeOH/CHCl₃. Fractions containing onespot by TLC (10% MeOH/CHCl₃ Rf=0.4) were pooled and evaporated to givethe pure product (400 mg).

3′-O-(13-D-5-fluoro-2′-deoxyuridinyl)-g-L-2′-deoxyuridine (27h)

The O-protected dimer, 26h (400 mg) was treated with 100 ml of methnolicammonia solution until the reaction is completed. The solvent wasstripped off in vacuo and the residue was purified on DEAE cellulose ionexchange column using gradient of NH₄CO₃ buffer from 0.02-0.2M. Purefractions were evaporated at 40° C. in high vacuo to dryness to give thepure product (175 mg).

NMR: (D₂O) δ=2.40 (m, 3H), 2.61 (m, 1H), 3.80 (m,2H), 4.10 (m, 2H), 4.18(m,2H), 4.55 (m, 1H), 4.80 (m, 1H), 5.85 (d, 1H), 6.30 (q, 2H), 7.85 (d,1H), 8.06 (d, 1H).

P³¹ NMR: (D₂O) δ=0.20(s)

5′-O-dimethoxytri-tyl-N⁴-benzoyl-2′-deoxy-β-L-cytidine (20i)

N⁴-benzoyl-2′-deoxy-β-L-cytidine (0.8 g, 2.42) was dissolved in 50 ml ofdry, distilled pyridine. To this solution was added 4,4′-dimethoxytritylchloride (3.0 g, 8.85 mmol) and 4-dimethylamino pyridine (DMAP) (60 mg,0.48 mmol). The mixture was stirred under an argon atmosphere for 16hours. After this time, the pyridine was stripped off in vacuo. Theresidue was dissolved in EtOAc (100 ml). The organic layer was washedwith water, saturated NaHCO₃, and brine. The organic layer was driedover Na₂SO₄, filtered and evaporated in vacuo to a residue which waspurified on a silica gel column using 10% MeOH/CHCl₃. Pure fractionswere pooled and evaporated to give the pure product as an off-white foam(1.49 g, (97% yield). Rf=0.48 in 10% MeOH/CHCl₃.

NMR: (CDCl₃-d₆) δ=2.3 (m, 1H), 2.75 (m, 2H), 3.42 (ddd, 2H), 3.80 (s,6H), 4.15 (q, 2H), 4.52 (m, 1H), 6.30 (t, 1H), 6.82 (dd, 4H), 7.2-7.6(m, Ar), 7.85 (d, 2H), 8.32 (d, 1H), 8.76 (br s, 1H).

5′-O-(dimethoxytrityl)-N⁴-benzoyl-2′-deoxy-β-L-cytidine-3′-N,N-diisopropylmethylphosphoramidite (21i)

The 5′-O-dimethoxytrityl-N⁴-benzoyl-2′-deoxy-p3-L-cytidine (0.6 g, 0.95mmol) was dissolved in anhydrous dichloromethane (50 ml).N,N-diisopropylethylamine (0.66 ml, 3.79 mmol) was added through aseptum, followed by chloro-N,N-diisopropylmethoxyphosphine (0.28 ml,1.42 mmol), under an argon atmosphere. The reaction was stirred for 30minutes. The solvent was evaporated and the residue was partitionedbetween an 80% EtOAc/triethylamine mixture and brine. The organic layerwas washed with saturated NaHCO₃ solution and brine. The organic residuewas evaporated to dryness and the residue was purified on a silica gelcolumn using a mixture of dichloromethane, EtOAc and triethylamine(60:30:10; Rf=0.8). The product was isolated quantitatively as a yellowfoam and it was used in the next step without further purification.

5′-O-dimethoxytrityl-3′-[O-(3′-O-acetyl)-β-D-5-fluoro-2′-deoxyuridinyl]-N⁴-benzoyl-2′-deoxy-β-L-cytidine(25U)

The 3′-O-acetyl)-β-D-5-fluoro-2′-deoxyuridine (0.23 g, 0.78 mmol) wasdissolved in dry acetonitrile (30 ml). Sublimed 1H-tetrazole (110 mg)was added and the mixture was stirred under an argon atmosphere for 15minutes. The solution of 21i (0.94 mmol), dissolved in 15 ml of dryacetonitrile was added via syringe to the reaction solution over 5minutes. The mixture was allowed to stir at room temperature for threehours. The acetonitrile was evaporated in vacuo to a residue. Thisresidue was triturated with a 70% EtOAc/ether mixture. The undissolvedtetrazole was filtered off and the filtrate was evaporated to give a dryyellow foam (0.73 g). This foam was used in the next step withoutfurther purification.

(3′-acetoxy-β-D-5-fluoro-2′-deoxyuridinyl)-N⁴-benzoyl-2′-deoxu-β-L-cytidinemethyl phosphonate ester (26i)

The dimer, 25i (0.73 g), was dissolved in 20 ml of THF and 4 ml ofpyridine containing 0.4 ml of water. Iodine crystals (100 mg) were addedand the contents of the loosely stoppered flask were allowed to stir for1 hour. Excess iodine was discharged by the addition of few drops ofsaturated sodium thiosulfate. The reaction mixture was then evaporatedto dryness. The crude product was EtOAc washed with saturated NaHCO₃solution and brine. The organic layer was dried over Na₂SO₄, filteredand evaporated in vacuo. The residue was dissolved in 25 ml of 80%acetic acid/water solution and was stirred until the reaction wascompleted. The solvent was evaporated and the residue was purified on asilica gel column, using 10-15% MeOH/CHCl₃. Fractions containing onespot by TLC (10% MeOH/CHCl₃ Rf=0.4) were pooled and evaporated to givethe pure product (108 mg).3′-O-(β-D-5-fluoro-2′-deoxyuridinyl)-2′-deoxy-O-L-cytidine (27i)

The O-protected dimer, 26i (108 mg) was treated with 100 ml of methnolicammonia solution until the reaction is completed. The solvent wasstripped off in vacuo and the residue was purified on DEAE cellulose ionexchange column using gradient of NH₄CO₃ buffer from 0.02-0.2M. Purefractions were evaporated at 40° C. in high vacuo to dryness to give thepure product (56 mg).

NMR: (D₂O) δ=2.30 (m, 3H), 2.55 (m, 1H), 3.80 (m, 2H), 4.05 (m, 2H),4.18 (m, 2H), 4.52 (m, 1H), 4.78 (m, 1H), 6.02 (d, 1H), 6.25 (m, 2H),7.80 (d, 1H), 8.04 (d, 1H).

P³¹ NMR: (D₂O) δ=0.05(s)

5′-O-dimethoxytrityl-3′-[O-(3′-O-acetyl)-N⁴-benzoyl-2′-deoxy-D-L-cytidinyl)-β-D-5-fluoro-2'deoxyuridine(25j)

The 3′-O-acetyl-β-D-5-fluoro-2′-deoxyuridine (0.25 g, 0.67 mmol) wasdissolved in dry acetonitrile (30 ml). Sublimed 1H-tetrazole (94 mg) wasadded and the mixture was stirred under an argon atmosphere for 15minutes. The solution of 21j (0.51 g, 0.67 mmol), dissolved in 15 ml ofdry acetonitrile was added via syringe to the reaction solution over 5minutes. The mixture was allowed to stir at room temperature for threehours. The acetonitrile was evaporated in vacuo to a residue. Thisresidue was triturated with a 70% EtOAc/ether mixture. The undissolvedtetrazole was filtered off and the filtrate was evaporated to give a dryyellow foam (0.49 9). This foam was used in the next step withoutfurther purification.

(3′-acetoxy-N⁴-benzoyl-2′-deoxy-(3-L-cytidinyl)-(β-D-5-fluoro-2′-deoxyuridinylcyanoethyl phosphonate ester (26i)

The dimer, 25j (0.49 g), was dissolved in 8 ml of THF and 2 ml ofpyridine containing 0.2 ml of water. Iodine crystals (30 mg) were addedand the contents of the loosely stoppered flask were allowed to stir for1 hour. Excess iodine was discharged by the addition of few drops ofsaturated sodium thiosulfate. The reaction mixture was then evaporatedto dryness. The crude product was EtOAc washed with saturated NaHCO₃solution and brine. The organic layer was dried over Na₂SO₄, filteredand evaporated in vacuo. The residue was dissolved in 20 ml of 80%acetic acid/water solution and was stirred until the reaction wascompleted. The solvent was evaporated and the residue was purified on asilica gel column, using 10-15% MeOH/CHCl₃. Fractions containing onespot by TLC (10% MeOH/CHCl₃ Rf=0.4) were pooled and evaporated to givethe pure product (188 mg).

3′-O-(2′-deoxy-β-L-cytidinyl)-β-D-5-fluoro-2′-deoxyuridine (27i)

The O-protected dimer, 26j (188 mg) was treated with 100 ml ofconcentrated ammonia solution until the reaction is completed. Thesolvent was stripped off in vacuo and the residue was purified on DEAEcellulose ion exchange column using gradient of NH₄CO₃ buffer from0.02-0.2M. Pure fractions were evaporated at 40° C. in high vacuo todryness to give the pure product (105 mg).

NMR: (D₂O) δ=2.30 (m, 3H), 2.50 (m, 1H), 3.80 (m, 2H), 4.05 (m, 2H),4.10 (m, 2H), 4.20 (m, 1H), 4.52 (m, 1H), 4.75 (m, 1H), 6.05 (d, 1H),6.29 (q, 2H), 7.89 (d, 1H), 9.03 (d, 1H).

P³¹NMR: (D₂O) δ=0.05(s)

5′-O-dimethoxytrityl-3′-[O-(3′-O-acetyl)-N⁴-benzoyl-2′-deoxy-α-L-cytidinyl)-β-D-5-fluoro-2′-deoxyuridine(25k)

The 3′-O-acetyl-β-D-5-fluoro-2′-deoxyuridine (0.19 g, 0.51 mmol) wasdissolved in dry acetonitrile (30 ml). Sublimed 1H-tetrazole (80 mg) wasadded and the mixture was stirred under an argon atmosphere for 15minutes. The solution of 21k (0.45 g, 0.61 mmol), dissolved in 15 ml ofdry acetonitrile was added via syringe to the reaction solution over 5minutes. The mixture was allowed to stir at room temperature for threehours. The acetonitrile was evaporated in vacuo to a residue. Thisresidue was triturated with a 70% EtOAc/ether mixture. The undissolvedtetrazole was filtered off and the filtrate was evaporated to give a dryyellow foam (0.42 g). This foam was used in the next step withoutfurther purification.

(3′-acetoxy-N⁴-benzoyl-2′-deoxy-α-L-cytidinyl)-β-D-5-fluoro-2′-deoxyuridinylcyanoethyl phosphonate ester (26k)

The dimer, 25k (0.42 g), was dissolved in 10 ml of THF and 2 ml ofpyridine containing 0.2 ml of water. Iodine crystals (45 mg) were addedand the contents of the loosely stoppered flask were allowed to stir for1 hour. Excess iodine was discharged by the addition of few drops ofsaturated sodium thiosulfate. The reaction mixture was then evaporatedto dryness. The crude product was EtOAc washed with saturated NaHCO₃solution and brine. The organic layer was dried over Na₂SO₄, filteredand evaporated in vacuo. The residue was dissolved in 25 ml of 80%acetic acid/water solution and was stirred until the reaction wascompleted. The solvent was evaporated and the residue was purified on asilica gel column, using 10-15% MeOH/CHCl₃. Fractions containing onespot by TLC (10% MeOH/CHCl₃ Rf=0.4) were pooled and evaporated to givethe pure product (125 mg).

3′-O-(2′-deoxy-α-L-cytidinyl)-(3-D-5-fluoro-2′-deoxyuridine (27k)

The O-protected dimer, 26k (125 mg) was treated with 100 ml ofconcentrated ammonia solution until the reaction is completed. Thesolvent was stripped off in vacuo and the residue was purified on DEAEcellulose ion exchange column using gradient of NH₄CO₃ buffer from0.02-0.2M). Pure fractions were evaporated at 40° C. in high vacuo todryness to give the pure product (40 mg).

NMR: (D₂O) δ=2.15 (m, 1H), 2.35 (m, 1H), 2.60 (m, 1H), 2.71 (m, 1H),3.81 (m, 2H), 3.97 (m, 2H), 4.22 (m, 1H), 4.52 (m, 2H), 6.02 (d, 1H),6.15 (dd, 1H), 6.28 (t, 1H), 7.87 (d, 1H), 8.03 (d, 1H).

P³¹NMR: (D₂O) δ=0.12(s)

EXAMPLE 5 Dimers Testing In Vitro in B16 Melanoma and P388 Leukemia andin Inhibition Assays Against 293 Processive Telomerase

The biological effects of the dimers were compared with those of themonomeric 5-FUdR on P388 leukemia and B16 melanoma cell lines and ininhibition assays against 293 processive telomerase. Telomerase is aDNA-processive enzyme that is not expressed in normal somatic cells butgenerally only in germ-line cells and fetal cells. In many types ofcancer cells, enzyme activity is reactivated, and others, telomeraseinhibitors can therefore serve as a valuable new class of antineoplasticagents. The results are shown in Table 2.

TABLE 1 Inhibition of Tumor Cell Growth and Telomerase Activity by5-FUdR Dinucleoside Monophosphates Telomerase Growth InhibitionInhibition IC₅₀ (nM) (Mean ± SEM) Compound P388 B16 % at 1 mN β-D FUDR2.8  28 0 α-L, β-D Dimer 0.41 2.45 84 ± 11 α-L FUDR NT 389,500 NT NT =Not Tested

The results indicate that the prototype dimers inhibit the growth ofmurine-cultured leukemic L1210 and melanoma B16 cells with great potency(some IC₅₀ values of less than 1 nM.) The IC50 values are several timesmore potent than FUdR. These results are unexpected and thus thesecompounds are truly unique.

The preliminary results of the telomerase inhibition are alsointriguing.

The α-L, β-D dimer inhibited the enzyme by 84% compared to control.

These data indicate that dimers containing an L-sugar have extremelyinteresting biological profiles and represent a novel class of potentantineoplastic agents. The activity profile of the L-dimers is differentfrom that of the parent monomeric drug β-D-5FUdR.

The biological effects of the dimers were compared with those of themonomeric 5-FUdR on P388 leukemia and B16 melanoma cell lines. Theresults are shown in Table 2.

TABLE 3 In Vitro Testing Data IC₅₀ (nM) CODE # COMPOUND P388 B16 L-102β-D-FUdR, α-L-FUdR 0.71 3.0 L-103 α-L-FUdR, β-D-FUdR 0.57 2.45 L-107α-L-dU, β-D-FUdR 7.0 219 L-108 α-L-FUdR, α-L-FUdR 22,200 52,200 L-109β-L-FUDR, β-L-FUdR 5860 45,900 L-110 β-L-FUdR, β-D-FUdR 2.0 6.3 L-111α-L-dC, β-D-FUdR 0.7 5.0 — β-D-FUdR 2.8 28

These data indicate that the dinucleoside monophosphate compoundscontaining β-D-5FUdR in conjunction with α-L or β-L-nucleosides showsuperior in vitro activities against murine P388 leukemia and B16melanoma cell lines, as is evidenced by lower IC₅₀ values. Thisindicates that such nucleoside dimers may indeed be acting by mechanismsdifferent from those of 5-FUdR or are metabolized and/or transporteddifferently from 5-FUdR.

EXAMPLE 6 Determination of Thymidylate Synthase Activity and ItsInhibition in Intact L1210 Leukemia Cells In Vitro

Thymidylate synthase is one suspected site of action of the compounds.Hence, the activity of selected compounds of the present invention,measured on thymidylate synthase activity measured in vitro, is areliable indicium of the behavior of these compounds in in vivo systems.

Mouse leukemia L1210 cells are harvested from the cell culture flasksand the cell concentration is determined. The cells are then resuspendedin the desired amount of the medium to give a stock concentration 5×10⁷cells/mL. Series of the dilution of the stock solution of the compoundsto be tested are prepared (concentrations are ranged from 10⁸ M to 10⁻³M). The solution of the compound to be tested in the desiredconcentration is pipetted into a microcentrifuge tube and incubated at37° C. using a shaking water bath. The reaction is started by additionof [5-³H]-2′-deoxycytidine (10 μL, concentration of the stocksolution—10⁻⁵ M) after a 30 or 60 min. preincubation with 80 μL of thecell suspension and allowed to proceed for 30 min. in a shaking waterbath at 37° C. The reaction is terminated by adding 100 μL of the 10%charcoal in 4% HC1O₄. The tubes are vigorously stirred by vortexing andthen centrifuged for 10 min. in a Beckman Microfuge. The radioactivityof a 100 μL of supernatant fraction from each tube is counted in aPackard Tri-Carb (model 2450 or 3255) liquid scintillation spectrometerusing a toluene based scintillation mixture. The release of tritium isexpressed as a percentage of the total amount of radioactivity added.IC₅₀ values determined from dose response curves represent theconcentration of inhibitors required for 50% inhibition of the releaseof tritium. Table 3 below shows the results of the analysis of tritiumrelease and determination of the IC₅₀.

TABLE 3 Inhibition of Tritium Sample Release, Code IC₅₀ (μM) 5FUdR 0.035L-102 0.035 L-103 0.035 L-107 >100 L-108 >100 L-109 >100 L-11O 6 L-111N/T

EXAMPLE 7 In Vivo Testing of Dimers in P388 Leukemia B16 Melanoma

A. Experimental

1. P388 Leukemia

B6D2F1 mice received i.p. inocula of P388 murine leukemia cells preparedby removing ascites fluid containing P388 cells from tumored B6D2FImice, centrifuging the cells, and then resuspending the leukemia cellsin saline. Mice received 1×10⁶ P388 cells i.p. on day 0. On day 1,tumored mice were treated with the dimers or vehicle control. The routeof drug administration was i.p. and the schedule selected was daily×5.The maximum tolerated doses (MTD) was 200 mg/kg for each dimer and wasdetermined in initial dose experiments in non-tumored mice. In theactual experiments, L-103 was given at 100 mg/kg and 50 mg/kg.

2. B16 Melanoma

B6D2F1 mice received i.p. inocula of B16 murine melanoma brei preparedfrom B16 tumors growing s.c. in mice (day 0). On day 1, tumored micewere treated with the dimers or vehicle control. The route of drugadministration was i.p. and the schedule selected was daily×5. Themaximum tolerated doses (MTD) was 200 mg/kg for each dimer and wasdetermined in initial dose experiments in non-tumored mice. In theactual experiments, L-103 was given at 100 mg/kg and 50 mg/kg.

3. Survival Standard

The mean survival times of all groups were calculated, and results areexpressed as mean survival of treated mice/mean survival of control mice(TIC)×100%. A TIC value of 150 means that the mice in the treated grouplived 50% longer than those of the control group; this is sometimesreferred to as the increase in life span, or ILS value.

In the P388 studies, mice that survive for 30 days are considered longterm survivors or cures while in B16; mice that survive for 60 days areconsidered long term survivors or cures. The universally acceptedcut-off for activity in both models, which has been used for years bythe NCl, is TIC=125. Conventional use of B 16 and P388 over the yearshas set the following levels of activity: T/C<125, no activity;T/C=125-150, weak activity; T/C=150-200, modest activity; T/C=200-300,high activity; T/C>300, with long term survivors; excellent, curativeactivity.

B. Results

1. P388 Leukemia

L-103 demonstrated modest activity in the P388 leukemia in mice at alldoses tested (Table 5). L-103 gave i.p. daily×5 at doses of 100 mg/kgand 50 mg/kg resulted in TIC values of 149 and 144 respectively.Fluorodeoxyuridine (FUdR) was used as the positive drug control in thisstudy; FUdR produced a T/C=164 in the P388 test (Table 4). All agentswere well-tolerated in this experiment; there was little or no bodyweight loss and no toxic deaths were recorded.

TABLE 4 L-103 vs. Murine P388 Leukemia Weight Change Group n Dose (Day7) T/C Control (10) 0.9% Saline +9.1% 100 L-103 (10) 100 mg/kg +1.5% 149L-103 (10) 50 mg/kg −1.6% 144 FUdR (10) 100 mg/kg −2.7% 164

2. B16 Melanoma

L-103 demonstrated modest activity against B16 melanoma implanted inmice (Table 6). L-103 (i.p.; daily×5) gave T/C values of 139 and 134respectively. The positive control drug FUdR resulted in modest efficacyin the B16 test; a T/C value of 135 was obtained (Table 5). All agentswere well-tolerated, with little or no weight loss; no drug-relateddeaths occurred.

TABLE 5 L-103 vs. Murine B16 Leukemia Weight Change Group n Dose (Day 7)T/C Control (10) 0.9% Saline +4.9% 100 L-103 (10) 100 mg/kg +2.3% 139L-103 (10) 50 mg/kg +6.3% 134 FUdR (10) 100 mg/kg −0.5% 135

L-103 demonstrated modest activity against both the P388 and B16experimental murine tumors at the two doses tested. L-103 wasapproximately as active as the positive control drug FUdR in the B16test, and was somewhat less active than FUdR in the P388 test.

From the foregoing, the significance of L-sugar-based α- andβ-enantiomeric nucleosides, nucleotides and their analogues asversatile, highly effective chemotherapeutic agents is apparent. Ourresults on derivatives of 5-FUdR show that L-5-FUdR-containing isomersare less toxic than those containing β-D-5FUdR, perhaps because they arenot phosphorylated or transported as the latter. We have found thatdimeric derivatives designed and prepared from α-L-5FUdR show verypotent activity against P388 leukemia cells and B16 melanoma cell lines,exceeding that of β-D-5FUdR. They appear to have unusual mechanisms ofaction, including inhibition of telomerase.

EXAMPLE 8 In Vivo Activity of Nucleoside Analogs

The different activity of the dimers is dependent on the structure ofthe second nucleoside.

The following plausible pathways for metabolic activation and/or mode ofaction of the dimer molecules tested are that:

(1) Dimer may act as a new chemical entity without hydrolysis of thephosphate or pro-phosphate bond between the two monomeric units;

(2) Hydrolysis to L-nucleoside and FUdR nucleotide may occur, in whichcase the dimer is a prodrug. The L-nucleoside is used for protection

and to increase the bioavailability of β-D-FUdR monophosphate.

It is also important to note that hydrolysis can take placeintracellularly as well as outside the cell.

In the last decade, monumental efforts have been directed toward thesynthesis of oligonucleotide analogs with altered phosphodiesterlinkage.

The goal was to improve the stability of duplex and triplex formation,to improve the cellular uptake and to decrease the rate of degradationof oligonucleotides by endo and exo nucleases which cleave thephosphodiester linkage. We selected one such chemical modification forour study. As a consequence, several dimers with phosphorothioatelinkage between two nucleosides were synthesized and tested. Thephosphorothioate comprises a sulfur-for-oxygen substitution atphosphorus of the phosphodiester linkage (for the structure of thecorresponding dimers see Appendix 1). It has been shown (M. Matsukura,K. Shinozuka, G. Zon, H. Mitsuya, M. Reitz, J. S. Cohen, L. M. Neckers,Proc. Natl. Acad. Sci. USA. 84, 7706 (1987)) that the S homologues aremore resistant to cellular nucleases and are readily taken up by cells.Several oligonucleotides of this type are currently in clinical studies(ISIS Pharmaceuticals and others).

EXAMPLE 8 Synthesis of N⁶-Benzoyl-3′-deoxy-β-D-adenosine

To a stirring solution of 3′-deoxyadenosine (2.0 g, 7.96 mmol) inpyridine (80 ml) chilled in an ice bath, ClSiMe₃ (5.0 ml, 39.8 mmol) wasadded dropwise and stirred for 30 minutes. Benzoyl chloride (3.7 ml,31.84 mmol) was then added dropwise and the reaction mixture was stirredat room temperature for two hours. This was cooled in an ice bath andwater (16 ml) was added dropwise. 15 minutes later concentrated NH₄0H(16 ml) was added to give a solution approximately 2M in ammonia. After30 minutes the solvent was evaporated and the residue was dissolved inwater and washed with ether. The water layer was concentrated and thecompound was crystallized from water as white solid (2.32 g. 82%).

EXAMPLE 9 Synthesis of N⁶-Benzoyl-5′-O-(dip-methoxytrityl)-3′-deoxy-β-D-adenosine

To a solution of compound N⁶-Benzoyl-3′deoxy-β-D-adenosine 1(2.32 g,6.53 mmol) in pyridine (100 ml) was added 4,4′-dimethoxytrityl chloride(3.32 g, 9.79 mmol) and DMAP (0.24 g, 1.96 mmol) and stirred at roomtemperature for 2 hours under argon. To complete the reaction,additional DMTCI (0.5 g) was added and stirred for another 2 hours. Thereaction was quenched with the addition of MeOH (5 ml) and the solventwas evaporated. The residue was dissolved in EtOAc, washed with water,NaHCO₃ and brine. After drying over Na₂SO₄, the EtOAc layer wasevaporated and the crude compound was purified on a silica gel columnusing 80% EtOAc/CHCl₃ as solvent to give pure compound 2 (4.33 g, 83%)as a white foam.

EXAMPLE 10 Synthesis of N⁶-Benzoyl-2′-O-acetoxy-(3-D-3′-deoxyadenosine

To a solution of N⁶-Benzoyl-5′-O-(dip-methoxytrityl)-3′-deoxy-β-D-adenosine (4.3 g, 6.58 mmol) in pyridine(100 ml) acetic anhydride (1 ml, 9.87 mmol), and DMAP (0.08 g, 0.65mmol) was added and stirred at room temperature for 15 minutes. Then thesolvent was evaporated and the residue was dissolved in EtOAc, washedwith water, NaHCO₃, brine and dried over NA₂SO₄. After the evaporationof EtOAc, then the crude material was dissolved in 80% AcOH (50 ml) andstirred at room temperature for one hour. Then the solvent wasevaporated and coevaporated with tolune and purified on a silica gelcolumn using 3-5% MeOH/CHCl₃ to give pureN⁶-Benzoyl-2′-O-acetoxy-β-D-3-deoxyadenosine (2.11 g, 81%) as a foam.

Example 11 Synthesis of β-L-dU, Cordycepin Dimer (L-150)

β-L-dU (1.0 g, 4.38 mmol) was dissolved in dry pyridine (50 ml), to thissolution was added 4,4′-dimethoxytrityl chloride (1.78 g, 5.25 mmol) andDMAP (0.1 g, 0.87 mmol). This was stirred under argon at roomtemperature for 2 hours and quenched with MeOH (5 ml). The solvent wasevaporated, the residue was dissolved in EtOAc, washed with water,NaHCO₃ and brine. After drying and evaporation of the solvent, the crudematerial was purified on a silica gel column using 60-80% EtOAc/CHCl₃ assolvent to give pure 5′-O-Dimethoxytrityl-β-L-2′-deoxyuridine (2.2 g,94.8%) as white foam.

Dimethoxytrityl-β-L-2′-deoxyuridine (1.5 g, 2.83 mmol) was dissolved inanhydrous dichloromethane (50 ml). N,N-diisopropylethylamine (2.0 ml,11.3 mmol) was added uner argon followed by2′-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.82 ml, 3.68 mmol).The reaction was stirred for 30 minutes and the solvent was evaporated.The residue was dissolved in 80% EtOAc/Et₃N (75 ml) and washed withwater, NaHCO₃ and brine. The organic layer was evaporated and purifiedon a short silica gel column using a mixture of EtOAc, CH₂Cl₂ and ET₃N(40:50:10) to give 5′-O-Dimethoxytrityl-β-L-2′-deoxyuridine-3′-N,N-diisopropylcyanoethyl phosphoramidite in quantitative yield.

5′-O-Dimethoxytrityl-3′-[O-(2′-O-acetyl)-N⁶-benzoyl-β-D-3′-deoxyadenosinyl]-2′-deoxy-β-L-uridine cyanoethyl phosphite ester (7).

To a solution of compound 5′-O-Dimethoxytrityl-β-L-2′-deoxyuridine-3′-N,N-diisopropylcyanoethyl phosphoramidite (2.83 mmol) in anhydrousacetonitrile (60 ml), N⁶-Benzoyl-2′-O-acetoxy-β-D-3-deoxyadenosine (1.12g, 2.83 mmol) in acetonitrile (40 ml) was added and stirred for 10minutes under argon. To this solution, sublimed 1H-tetrazole (0.6 g, 8.5mmol) was added and stirred over night. The solvent was evaporated andthe residue was triturated with 70% EtOAc/ether and filtered. Thefiltrate was evaporated to give5′-O-Dimethoxytrityl-3′-[O-(2′-O-acetyl)-N⁶-benzoyl-β-D-3′-deoxyadenosinyl]-2′-deoxy-β-L-uridine cyanoethyl phosphite ester as a foamand this was used in the next step without further purification.

The 5′-O-Dimethoxytrityl-3′-[O-(2′-O-acetyl)-N⁶-benzoyl-β-D-3′-deoxyadenosinyl]-2′-deoxy-β-L-uridine cyanoethyl phosphite ester wasdissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml). Iodinecrystals (1.0 g) were added portion wise until the iodine colorpersists. The reaction mixture was stirred for another 15 minutes andthe excess iodine was removed by the addition of saturated sodiumthiosulfate. The solvent was evaporated and the residue was dissolved inEtOAc and washed with water, NaHCO₃ and brine. EtOAc layer wasevaporated and the residue was dissolved in 80% acetic acid/watersolution (40 ml) and stirred for 1 hour. Then the solvent was evaporatedand the crude product was purified on a silica gel column using 8-15%MeOH/CHCl₃ as solvent to give pure(2′-Acetoxy-N⁶-benzoyl-3′-deoxy-β-D-adenosinyl)-β-L-2′-deoxyuridinylcyanoethyl phosphate ester (0.75 g) as a foam.

The dimer(2′-Acetoxy-N⁶-benzoyl-3′-deoxy-β-D-adenosinyl)-β-L-2′-deoxyuridinylcyanoethyl phosphate ester (0.75 g) was treated with ammoniun hydroxidesolution (100 ml) over night. The solvent was evaporated and the residuewas purified on DEAE Cellulose ion exchange column using gradient ofNH₄HCO₃ buffer (0.05-0.2M). The pure fractions were collected andlyophillized to give pure3′-O-(3′-deoxy-β-D-adenosinyl)-β-L-2′-deoxyuridine (L-150)(0.486 g) aswhite solid.

EXAMPLE 12 Synthesis of β-L-dA, Cordycepin dimer (L-151)

To a stirring solution of 2′-deoxy-β-L-adenosine (2.05 g, 8.16 mmol) inpyridine (75 ml) chilled in an ice bath, ClSiMe₃ (5.17 ml, 40.8 mmol)was added dropwise and stirred for 30 minutes. Benzoyl chloride (4.7 ml,40.8 mmol) was then added dropwise and the reaction mixture was stirredat room temperature for two hours. This was cooled in an ice bath andwater (15 ml) was added dropwise. 15 minutes later concentrated NH₄0H(15 ml) was added to give a solution approximately 2 M in ammonia. After30 minutes the solvent was evaporated and the residue was dissolved inwater and washed with ether. The water layer was concentrated and theN⁶-benzoyl-2′-deoxy-β-L-adenosine was crystallized from water as whitesolid (2.48 g, 85.8%).

To a solution of N⁶-benzoyl-2′-deoxy-β-L-adenosine (2.48 g, 6.98 mmol)in pyridine (100 ml) was added 4,4′-dimethoxy trityl chloride (3.55 g,10.47 mmol) and DMAP (0.25 g, 2.09 mmol) and stirred at room temperaturefor 2 hours under argon. To complete the reaction, additional DMTCI (1.3g) was added and stirred for another 2 hours. The reaction was quenchedwith the addition of MeOH (5 ml) and the solvent was evaporated. Theresidue was dissolved in EtOAc, washed with water, NaHCO₃ and brine.After drying over Na₂SO₄, the EtOAc layer was evaporated and the crudecompound was purified on a silica gel column using 3-5% MeOH/CHCl, assolvent to give pure N⁶-benzoyl-5′-O-(di-p-methoxytrityl)-2′-deoxy-β-L-adenosine (3.42 g, 74.5%) as pale yellow foam.

N⁶-benzoyl-5′-O-(di-p-methoxy trityl)-2′-deoxy-β-L-adenosine (1.64 g,2.5 mmol) was dissolved in anhydrous dichloromethane (50 ml).N,N-diisopropylethylamine (1.75 ml, 10.0 mmol) was added under argonfollowed by 2′-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.8 ml,3.25 mmol). The reaction was stirred for 30 minutes and the solvent wasevaporated. The residue was dissolved in 80% EtOAc/Et₃N (75 ml) andwashed with water, NaHCO₃ and brine. The organic layer was evaporatedand purified on a short silica gel column using a mixture of EtOAc,CH₂CL₂ and Et₃N (40:50: 10) to giveN′-Benzoyl-5′-O-(dimethoxytrityl)-β-L-2′-deoxyadenosine-3′-N,N-diisopropylcyanoethylphosphoramidite in quantitative yield.

To a solution ofN⁶-Benzoyl-5′-O-(dimethoxytrityl)-β-L-2′-deoxyadenosine-3′-N,N-diisopropylcyanoethylphosphoramidite (2.5 mmol) in anhydrous acetonitrile (60 ml),N⁶-Benzoyl-2′-O-acetoxy-β-D-3′-deoxyadenosine (0.94 g, 2.36 mmol) inacetonitrile (40 ml) was added and stirred for 10 minutes under argon.To this solution, sublimed 1H-tetrazole (0.5 g, 7.2 mmol) was added andstirred over night. The solvent was evaporated and the residue wastriturated with 70% EtOAc/ether and filtered. The filtrate wasevaporated to giveN⁶-Benzoyl-5′-O-(dimethoxytrityl)-3′-[O-(2′-O-acetyl)-N⁶-benzoyl-β-D-3-deoxyadenosinyl]-2′-deoxy-β-L-adenosine cyanoethyl phosphite ester as a foamand this was used in the next step without further purification.

The dimerN⁶-Benzoyl-5′-O-(dimethoxytrityl)-3′-[O-(2′-O-acetyl)-N⁶-benzoyl-β-D-3-deoxyadenosinyl]-2′-deoxy-β-L-adenosine cyanoethyl phosphite was dissolved inTHF (24 ml), pyridine (6 ml) and water (0.6 ml). Iodine crystals (0.63g) were added portion wise until the iodine color persists. The reactionmixture was stirred for another 15 minutes and the excess iodine wasremoved by the addition of saturated sodium thiosulfate. The solvent wasevaporated and the residue was dissolved in EtOAc and washed with water,NaHCO₃ and brine. EtOAc layer was evaporated and the residue wasdissolved in 80% acetic acid/water solution (50 ml) and stirred for 1hour. Then the solvent was evaporated and the crude product was purifiedon a silica gel column using 5-10% MeOH/CHCl₃ as solvent to give pure(2′-Acetoxy-N⁶-benzoyl-3′-deoxy-β-D-adenosinyl)-N⁶-benzoyl-β-L-2′-deoxyadenosinylcyanoethyl phosphate ester (0.97 g) as a foam.

The dimer(2′-Acetoxy-N⁶-benzoyl-3′-deoxy-β-D-adenosinyl)-N⁶-benzoyl-β-L-2′-deoxyadenosinylcyanoethyl phosphate ester (0.97 g) was treated with ammonium hydroxidesolution (100 ml) over night. The solvent was evaporated and the residuewas purified on DEAE Cellulose ion exchange column using gradient ofNH₄HCO₃ buffer (0.05-0.2M). The pure fractions were collected andlyophillized to give pure compound3′-O-(3′-deoxy-β-D-adenosinyl)-β-L-2′-deoxyadenosine (15) (L-151) (0.55g) as white solid.

EXAMPLE 13 Synthesis of α-L-dU, Cordycepin Dimer (L-152)

α-L-dU (1.04 g, 4.5 mmol) was dissolved in dry pyridine (50 ml), to thissolution was added 4, 4′-dimethoxytrityl chloride (2.4 g, 6.86 mmol) andDMAP (0.11 g, 0.91 mmol). This was stirred under argon at roomtemperature for 2 hours and quenched with MeOH (5mL). The solvent wasevaporated, the residue was dissolved in EtOAc, washed with water,NaHCO₃ and brine. After drying and evaporation of the solvent, the crudematerial was purified on a silica gel column using 3-5% MeOH/CHCl₃ assolvent to give pure 5′-O-Dimethoxytrityl-α-L-2′-deoxyuridine (2.4 g.,99%) as white foam.

5′-O-Dimethoxytrityl-α-L-2′-deoxyuridine (1.73 g, 3.26 mmol) wasdissolved in anhydrous dichloromethane (30 ml),N,N-diisopropylethylamine (2.3 ml, 13.04 mmol) was added under argonfollowed by 2′-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.95 ml,4.23 mmol). The reaction was stirred for 30 minutes and the solvent wasevaporated. The residue was dissolved in 80% EtOAc/Et₃N (75 ml) andwashed with water, NaHCO₃ and brine. The organic layer was evaporatedand purified on a short silica gel column using a mixture of EtOAc,CH₂CL₂ and Et₃N (40:50:10) to give5′-O-Dimethoxytrityl-α-L-2′-deoxyuridine-3′-N, N-diisopropylcyanoethylphosphoramidite (2.26 g, 95%) as a foam.

To a solution of 5′-O-Dimethoxytrityl-α-L-2′-deoxyuridine-3′-N,N-diisopropylcyanoethyl phosphoramidite (2.26 g, 3.09 mmol) in anhydrousacetonitrile (60 ml), N⁶-Benzoyl-2′-O-acetoxy-β-D-3′-deoxyadenosine(1.35 g, 3.4 mmol) in acetonitrile (40 ml) was added and stirred for 10minutes under argon. To this solution, sublimed 1H-tetrazole (0.65 g,8.5 mmol) was added and stirred over night. The solvent was evaporatedand the residue was triturated with 70% EtOAc/ether and filtered. Thefiltrate was evaporated to give5′-O-Dimethoxytrityl-3′-[O-(2′-O-acetyl)-N⁶-benzoyl-β-D-3′-deoxyadenosinyl]-2′-deoxy-α-L-uridinecyanoethyl phosphite ester as a foam and this was used in the next stepwithout further purification.

The dimer5′-O-Dimethoxytrityl-3′-[O-(2′-O-acetyl)-N⁶-benzoyl-β-D-3′-deoxyadenosinyl]-2′-deoxy-α-L-uridinecyanoethyl phosphite ester was dissolved in THF (24 ml), pyridine (6 ml)and water (0.6 ml). Iodine crystals (0.7 g) were added portion wiseuntil the iodine color persists. The reaction mixture was stirred foranother 15 minutes and the excess iodine was removed by the addition ofsaturated sodium thiosulfate. The solvent was evaporated and the residuewas dissolved in EtOAc and washed with water, NaHCO₃ and brine. EtOAclayer was evaporated and the residue was dissolved in 80% aceticacid/water solution (50 ml) and stirred for 1 hour. Then the solvent wasevaporated and the crude product was purified on a silica gel columnusing 8-15% MeOH/CHCl₃ as solvent to give pure(2′-Acetoxy-N⁶-Benzoyl-3′-deoxy-β-D-adenosinyl)-α-L-2′-deoxyuridinylcyanoethyl phosphate ester (1.29 g) as a foam.

3′O-(3′-deoxy-β-D-adenosinyl)-α-L-2′-deoxyuridine (6) (L-152)

The dimer(2′-Acetoxy-N⁶-Benzoyl-3′-deoxy-β-D-adenosinyl)-α-L-2′-deoxyuridinylcyanoethyl phosphate ester (1.29 g) was treated with ammonium hydroxidesolution (100 ml) over night, The solvent was evaporated and the residuewas purified on DEAE Cellulose ion exchange column using gradient ofNH₄HCO₃ buffer (0.05-0.2M). The pure fractions were collected andlyophillized to give pure3′-O-(3′-deoxy-β-D-adenosinyl)-α-L-2′-deoxyuridine (L-152) (0.856 g) aswhite solid.

EXAMPLE 14 Synthesis of β-L-dC, Cordycepin Dimer (L-153)

To a solution of β-L-dCBz (1.7 g, 5.22 mmol) in pyridine (100 ml) wasadded 4,4′-dimethoxy trityl chloride (2.65 g, 7.83 mmol) and DMAP (0.13g, 1.04 mmol) and stirred at room temperature for 2 hours under argon.

To complete the reaction, additional DMTCI (0.9 g) was added and stirredfor another 2 hours. The reaction was quenched with the addition of MeOH(5 ml) and the solvent was evaporated. The residue was dissolved inEtOAc, washed with water, NaHCO₃ and brine. After drying over Na₂SO₄,the EtOAc layer was evaporated and the crude compound was purified on asilica gel column using 3-5% MeOH/CHCl₃ as solvent to give pureN⁴-Benzoyl-5′-O-(di-p-methoxytrityl)-2′-deoxy-β-L-cytidine (2.98 g, 90%)as pale yellow foam.

N⁴-Benzoyl-5′-O-(dimethoxytrityl)-β-L-2′-deoxycytidine-3′-N,N-diisopropylcyanoethylphosphoramidite (3)

N⁴-Benzoyl-5′-O-(di-p-methoxy trityl)-2′-deoxy-β-L-cytidine (1.7 g, 2.68mmol) was dissolved in anhydrous dichloromethane (50 ml),N,N-diisopropylethylamine (1.9 ml, 10.72 mmol) was added under argonfollowed by 2′-cyanoethyl-N, N-diisopropylchlorophosphoramidite (0.85ml, 3.5 mmol). The reaction was stirred for 30 minutes and the solventwas evaporated. The residue was dissolved in 80% EtOAc/Et₃N (75 ml) andwashed with water, NaHCO₃ and brine. The organic layer was evaporatedand purified on a short silica gel column using a mixture of EtOAc,CH₂Cl₂ and Et₃N (30:60:10) to giveN⁴-Benzoyl-5′-O-(dimethoxytrityl)-β-L-2′-deoxycytidine-3′-N,N-diisopropylcyanoethylphosphoramidite (2.06 g, 92%) as a foam.

To a solution ofN⁴-Benzoyl-5′-O-(dimethoxytrityl)-β-L-2′-deoxycytidine-3′-N,N-diisopropylcyanoethylphosphoramidite (2.06 g, 2.47 mmol) in anhydrous acetonitrile (100ml),N⁶-benzoyl-2′-O-acetoxy-β-D-3′-deoxyadenosine (1.08 g, 2.72 mmol) inacetonitrile (40 ml) was added and stirred for 10 minutes under argon.To this solution, sublimed 1H-tetrazole (0.52 g, 7.4 mmol) was added andstirred over night. The solvent was evaporated and the residue wastriturated with 70% EtOAc/ether and filtered.

The filtrate was evaporated to giveN⁴-Benzoyl-5′-O-dimethoxytrityl-3′-[O-(2′-O-acetyl)-N⁶-benzoyl-β-D-3′-deoxyadenosinyl]-2′-deoxy-β-L-cytidine cyanoethyl phosphite ester as a foamand this was used in the next step without further purification. ThedimerN⁴-Benzoyl-5′-O-dimethoxytrityl-3′-[O-(2′-O-acetyl)-N⁶-benzoyl-β-D-3′-deoxyadenosinyl]-2′-deoxy-β-L-cytidine cyanoethyl phosphite ester wasdissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml). Iodinecrystals (0.55 g) were added portion wise until the iodine colorpersists. The reaction mixture was stirred for another 15 minutes andthe excess iodine was removed by the addition of saturated sodiumthiosulfate.

The solvent was evaporated and the residue was dissolved in EtOAc andwashed with water, NaHCO₃ and brine. EtOAc layer was evaporated and theresidue was dissolved in 80% acetic acid/water solution (50 ml) andstirred for 1 hour. Then the solvent was evaporated and the crudeproduct was purified on a silica gel column using 5-10% MeOH/CHCl₃ assolvent to give pure compound(2′-Acetoxy-N⁶-benzoyl-3′-deoxy-β-D-adenosinyl)-N⁴-Benzoyl-β-L-2′-deoxycitydinylcyanoethyl phosphate ester (1.46 g) as afoam.

3′-O-(3′-deoxy-β-D-adenosinyl)-β-L-2′-deoxycytidine (6) (L-153)

The dimer(2′-Acetoxy-N⁶-benzoyl-3′-deoxy-β-D-adenosinyl)-N⁴-Benzoyl-β-L-2′-deoxycitydinylcyanoethyl phosphate ester (1.46 g) was treated with ammoniun hydroxidesolution (100 ml) over night. The solvent was evaporated and the residuewas purified on DEAE Cellulose ion exchange column using gradient ofNH₄HCO₃ buffer (0.05-0.2M). The pure fractions were collected andlyophillized to give pure3′-O-(3′-deoxy-β-D-adenosinyl)-β-L-2′-deoxycytidine (L-153) (0.81 g) aswhite solid.

EXAMPLE 15 Synthesis of α-L-dC, Cordycepin dimer (L-154)

To a solution of α-L-dC (1.6 g, 4.88 mmol) in pyridine (100 ml) wasadded 4, 4′-dimethoxy trityl chloride (2.43 g, 7.2 mmol) and DMAP (0.13g, 1.04 mmol) and stirred at room temperature for 2 hours under argon.To complete the reaction, additional DMTCI (1.0 g) was added and stirredfor another 2 hours. The reaction was quenched with the addition of MeOH(5 ml) and the solvent was evaporated. The residue was dissolved inEtOAc, washed with water NaHCO₃ and brine. After drying over Na₂SO₄, theEtOAc layer was evaporated and the crude compound was purified on asilica gel column using 3-5% MeOH/CHCl₃ as solvent to give pureN⁴-Benzoyl-5′-O-(di-p-methoxytrityl)-2′-deoxy-α-L-cytidine (2.34 g, 76%)as pale yellow foam.

N⁴-Benzoyl-5′-O-(di-p-methoxy trityl)-2′-deoxy-α-L-cytidine (1.84 g, 2.9mmol) was dissolved in anhydrous dichloromethane (50 ml).N,N-diisopropylethylamine (2.0 ml, 11.6 mmol) was added under argonfollowed by 2′-cyanoethyl-N, N-diisopropylchlorophosphoramidite (0.85ml, 3.5 mmol).

The reaction was stirred for 30 minutes and the solvent was evaporated.The residue was dissolved in 80% EtOAc/Et₃N (75 ml) and washed withwater, NaHCO₃ and brine. The organic layer was evaporated and purifiedon a short silica gel column using a mixture of EtOAc, hexane and Et₃N(50:40:10) to giveN⁴-Benzoyl-5′-O-(dimethoxytrityl)-α-L-2′-deoxycytidine-3′-N,N-diisopropylcyanoethylphosphoramidite (2.01 g, 83%) as a foam.

To a solution of compoundN⁴-Benzoyl-5′-O-(dimethoxytrityl)-α-L-2′-deoxycytidine-3′-N,N-diisopropylcyanoethylphosphoramidite (2.01 g, 2.4 mmol) in anhydrous acetonitrile (100 ml),N⁶-Benzoyl-2′-O-acetoxy-β-D-3′-deoxyadenosine (1.05 g, 2.65 mmol) inacetonitrile (40 ml) was added and stirred for 10 minutes under argon.To this solution, sublimed 1-H-tetrazole (0.5 g, 7.2 mmol) was added andstirred over night. The solvent was evaporated and the residue wastriturated with 70% EtOAc/ether and filtered. The filtrate wasevaporated to giveN⁴-Benzoyl-5′-O-(dimethoxytrityl)-3′-[O-(2′-acetyl)-N⁶-Benzoyl-β-D-3′-deoxyadenosinyl]-2′-deoxy-α-L-cytidine cyanoethyl phosphite ester as a foamand this was used in the next step without purification.

The dimerN⁴-Benzoyl-5′-O-(dimethoxytrityl)-3′-[O-(2′-acetyl)-N⁶-Benzoyl-β-D-3′-deoxyadenosinyl]-2′-deoxy-α-L-cytidine cyanoethyl phosphite ester wasdissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml). Iodinecrystals (0.5 g) were added portion wise until the iodine colorpersists. The reaction mixture was stirred for another 15 minutes andthe excess iodine was removed by the addition of saturated sodiumthiosulfate. The sulfate was evaporated and the residue was dissolved inEtOAc and washed with water, NaHCO₃ and brine. EtOAc layer wasevaporated and the residue was dissolved in 80% acetic acid/watersolution (50 ml) and stirred for 1 hour. Then the solvent was evaporatedand the crude product was purified on a silica gel column using 5-10%MeOH/CHCl₃ as solvent to give pure(2′-Acetoxy-N⁶-Benzoyl-3′-deoxy-β-D-adenosinyl)-N⁴-Benzoyl-α-L-2-deoxycitydinylcyanoethyl phosphate ester (1.8 g) as a foam.

The dimer(2′-Acetoxy-N⁶-Benzoyl-3′-deoxy-β-D-adenosinyl)-N⁴-Benzoyl-α-L-2-deoxycitydinylcyanoethyl phosphate ester (1.8 g) was treated with ammonium hydroxidesolution (100 ml) over night, The solvent was evaporated and the residuewas purified on DEAE Cellulose ion exchange column using gradient ofNH₄HCO₃ buffer (0.05-0.2M). The pure fractions were collected andlyophillized to give pure3′-O-(3′-deoxy-β-D-adenosinyl)-α-L-2′-deoxycytidine (L-154) (1.08 g) aswhite solid.

EXAMPLE 16 Synthesis of α-L-dA, Cordycepin Dimer (L-155)

To a stirring solution of 2′-deoxy-α-L-adenosine (2.05 g, 8.16 mmol) inpyridine (75 ml) chilled in an ice bath, ClSiMe₃ (5.17 ml, 40.8 mmol)was added dropwise and stirred for 30 minutes. Benzoyl chloride (4.7 ml,40.8 mmol) was then added dropwise and the reaction mixture was stirredat room temperature for two hours. This was cooled in an ice bath andwater (15 ml) was added dropwise, 15 minutes later concentrated NH₄0H(15 ml) was added to give a solution approximately 2M in ammonia. After30 minutes the solvent was evaporated and the residue was dissolved inwater and washed with ether. The water layer was concentrated and theN⁶-Benzoyl-2′-deoxy-α-L-adenosine crystallized from water as white solid(2.48 g, 85.8%).

To a solution of compound N⁶-Benzoyl-2′-deoxy-α-L-adenosine (2.48 g,6.98 mmol) in pyridine (100 ml) was added 4, 4′-dimethoxy tritylchloride (3.55 g, 10.47 mmol) and DMAP (0.25 g, 2.09 mmol) and stirredat room temperature for 2 hours under argon. To complete the reaction,additional DMTCI (1.3 g) was added and stirred for another 2 hours. Thereaction was quenched with the addition of MeOH (5 ml) and the solventwas evaporated. The residue was dissolved in EtOAc, washed with water,NaHCO₃ and brine. After drying over Na₂SO₄, the EtOAc layer wasevaporated and the crude compound was purified on a silica gel columnusing 3-5% MeOH/CHCl₃ as solvent to give pureN⁶-Benzoyl-5′-O-(di-p-methoxy trityl)-2′-deoxy-α-L-adenosine (3.42 g,74.5%) as pale yellow foam.

N⁶-Benzoyl-5′-O-(di-p-methoxy trityl)-2′-deoxy-α-L-adenosine (1.64 g,2.5 mmol) was dissolved in anhydrous dichloromethane (50 ml).N,N-diisopropylethylamine (1.75 ml, 10.0 mmol) was added under argonfollowed by 2′-cyanoethyl-N, N-diisopropylchlorophosphoramidite (0.8 ml,3.25 mmol). The reaction was stirred for 30 minutes and the solvent wasevaporated. The residue was dissolved in 80% EtOAc/Et₃N (75 ml) andwashed with water, NaHCO₃ and brine. The organic layer was evaporatedand purified on a short silica gel column using a mixture of EtOAc,CH₂Cl₂ and Et₃N (40:50: 10) to giveN⁶-Benzoyl-5′-O-(dimethoxytrityl)-α-L-2′-deoxyadenosine-3′-N,N-diisopropylcyanoethylphosphoramidite in quantitative yield.

To a solution ofN⁶-Benzoyl-5′-O-(dimethoxytrityl)-α-L-2′-deoxyadenosine-3′-N,N-diisopropylcyanoethylphosphoramidite(2.5 mmol) in anhydrous acetonitrile (60 ml),N⁶-Benzoyl-3′-O-acetoxy-β-D-2′-deoxyadenosine (0.94 g, 2.36 mmol) inacetonitrile (40 ml) was added and stirred for 10 minutes under argon.To this solution, sublimed 1H-tetrazole (0.5 g, 7.2 mmol) was added andstirred overnight. The solvent was *4′ evaporated and the residue wastriturated with 70% EtOAct ether and filtered. The filtrate wasevaporated to giveN⁶-Benzoyl-5′-O-dimethoxytrityl-3′-[O-(2′-)-acetyl)-N⁶-benzoyl-β-D-3′-deoxyadenosinyl]-2′-deoxy-α-L-adenosine cyanoethyl phosphite ester as a foamand this was used in the next step without further purification.

The dimerN⁶-Benzoyl-5′-O-dimethoxytrityl-3′-[O-(2′-)-acetyl)-N⁶-benzoyl-β-D-3′-deoxyadenosinyl]-2′-deoxy-α-L-adenosine cyanoethyl phosphite ester wasdissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml). Iodinecrystals (0.63 g) were added portion wise until the iodine colorpersists. The reaction mixture was stirred for another 15 minutes andthe excess iodine was removed by the addition of saturated sodiumthiosulfate. The solvent was evaporated the residue was dissolved inEtOAc and washed with water, NaHCO₃ and brine. EtOAc layer wasevaporated and the residue was dissolved in 80% acetic acid/watersolution (50 ml) and stirred for 1 hour. Then the solvent was evaporatedand the crude product was purified on a silica gel column using 5-10%MeOH/CHCl₃ as solvent to give pure compound(2′-Acetoxy-N⁶-benzoyl-3′-deoxy-β-D-adenosinyl)-N⁶-benzoyl-α-L-2′-deoxyadenosinylcyanoethyl phosphate ester (0.97 g) as a foam.

The dimer(2′-Acetoxy-N⁶-benzoyl-3′-deoxy-β-D-adenosinyl)-N⁶-benzoyl-α-L-2′-deoxyadenosinylcyanoethyl phosphate ester(0.97 g) was treated with ammonium hydroxidesolution (100 ml) overnight. The solvent was evaporated and the residuewas purified on DEAE Cellulose ion exchange column using gradient ofNH₄HCO₃ buffer (0.05-0.2M). The pure fractions were collected andlyophillized to give pure3′-O-(3′-deoxy-β-D-adenosinyl)-β-L-2′-deoxyadenosine (0.55 g)(L-1 55) aswhite solid.

EXAMPLE 17

Synthesis of β-L-dA, β-D-dA (L-210)

To a stirring solution of 2′-deoxy-β-L-adenosine (2.05 g, 8.16 mmol) inpyridine (75 ml) chilled in an ice bath, ClSiMe₃ (5.17 ml, 40.8 mmol)was added dropwise and stirred for 30 minutes. Benzoyl chloride (4.7 ml,40.8 mmol) was then added dropwise and the reaction mixture was stirredat room temperature for two hours. This was cooled in an ice bath, andwater (15 ml) was added dropwise. 15 minutes later concentrated NH₄OH(15 ml) was added to give a solution approximately 2M in ammonia. After30 minutes, the solvent was evaporated and the residue was dissolved inwater and washed with ether. The water layer was concentrated, and theN⁶-Benzoyl-2′-deoxy-β-L-adenosine was crystallized from water as whitesolid (2.48 g, 85.8%).

To a solution of N⁶-Benzoyl-2′-deoxy-β-L-adenosine(2.48 g, 6.98 mmol) inpyridine (100 ml) was added 4,4′-dimethoxy trityl chloride (3.55 g,10.47 mmol) and DMPA (0.25 g, 2.09 mmol) and stirred at room temperaturefor 2 hours under argon. To complete the reaction, additional DMTC1 (1.3g) was added and stirred for another 2 hours. The reaction was quenchedwith the addition of MeOH (5 ml), and the solvent was evaporated. Theresidue was dissolved in EtOAc, washed with water, NaHCO₃ and brine.After drying over Na₂SO₄, the EtOAc layer was evaporated and the crudecompound was purified on a silica gel column using 3/5% MeOH/CHC1₃ assolvent to give pure N⁶-Benzoyl-5′-O-(di-p-methoxytrityl)-2′-deoxy-β-L-adenosine (3.42 g, 74.5%) as pale yellow foam.

N⁶-Benzoyl-5′-O-(di-p-methoxy trityl)-2′-deoxy-β-L-adenosine(1.71 g,2.61 mmol) was dissolved in anhydrous dichloromethane (50 ml).N,N-diisopropylethylamine (1.8 ml, 10.34 mmol) was added under argonfollowed by 2′-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.0 ml,4.47 mmol). The reaction was stirred for 30 minutes, and the solvent wasevaporated. The residue was dissolved in 80% EtOAc/Et₃N (75 ml) andwashed with water, NaHCO₃ and brine. The organic layer was evaporatedand purified on a short silica gel column using a mixture of EtOAc,hexane and Et₃N (50:40:10) to giveN⁶-Benzoyl-5′-O-(dimethoxytrityl)-β-L-2′-deoxyadenosine-3′-N,N-diisopropylcyanoethyl phosphoramidite(1.87 g, 84%) as a foam.

To a solution ofN⁶-Benzoyl-5′-O-(dimethoxytrityl)-β-L-2′-deoxyadenosine-3′-N,N-diisopropylcyanoethylphosphoramidite(1.87 g, 2.18 mmol) in anhydrous acetonitrile (60 ml),N⁶-Benzoyl-3′-O-acetoxy-β-D-2′-deoxyadenosine (0.95 g, 2.4 mmol) inacetonitrile (40 ml) was added and stirred for 10 minutes under argon.To this solution, sublimed 1H-tetrazole (0.46 g, 6.6 mmol) was added andstirred overnight. The solvent was evaporated, and the residue wastriturated with 70% EtOAc/ether and filtered. The filtrate wasevaporated to giveN⁶-Benzoyl-5′-O-dimethoxytrityl-3′-[O-(3′-O′acetyl)-N⁶-benzoyl-β-D-2′-deoxyadenosinyl]-2′-deoxy-β-L-adenosine cyanoethyl phosphite ester as a foam,and this was used in the next step without further purification.

The dimerN⁶-Benzoyl-5′-O-dimethoxytrityl-3′-[O-(3′-O′acetyl)-N⁶-benzoyl-β-D-2′-deoxyadenosinyl]-2′-deoxy-β-L-adenosine cyanoethyl phosphite ester wasdissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml). Iodinecrystals (0.5 g) were added portion wise until the iodine colorpersists. The reaction mixture was stirred for another 15 minutes, andthe excess iodine was removed by the addition of saturated sodiumthiosulfate. The solvent was evaporated, and the residue was dissolvedin EtOAc and washed with water, NaHCO₃ and brine. EtOAc layer wasevaporated, and the residue was dissolved in 80% acetic acid/watersolution (50 ml) and stirred for 1 hour. Then the solvent wasevaporated, and the crude product was purified on a silica gel columnusing 5-10% MeOH/CHC1₃ as solvent to give pure compound(3′-Acetoxy-N⁶-benzoyl-2′-deoxy-β-D-adenosinyl)-N⁶-benzoyl-β-L-2′-deoxyadenosinylcyanoethyl phosphate ester (0.13 g) as a foam.

The dimer(3′-Acetoxy-N⁶-benzoyl-2′-deoxy-β-D-adenosinyl)-N⁶-benzoyl-β-L-2′-deoxyadenosinylcyanoethyl phosphate ester(1.3 g) was treated with ammonium hydroxidesolution (100 ml) overnight. The solvent was evaporated, and the residuewas purified on DEAE Cellulose ion exchange column using gradient ofNH₄HCO₃ buffer (0.05-0.2M). The pure fractions were collected andlyophillized to give pure3′-O-(2′-deoxy-β-D-adenosinyl)-β-L-2′-deoxyadenosine (L-210) (0.640 g)as white solid.

All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

One skilled in the art readily appreciates that the patent invention iswell adapted to carry out the objectives and obtain the ends andadvantages mentioned as well as those inherent therein. Dimers,pharmaceutical compositions, treatments, methods, procedures andtechniques described herein are presently representative of thepreferred embodiments and are intended to be exemplary and are notintended as limitations of the scope. Changes therein and other useswill occur those skilled in the art which are encompassed within thespirit of the invention or defined by the scope of the pending claims.

What is claimed is:
 1. A pharmaceutical composition comprised of anucleoside dimer having the formula: R₁—X—R₂ wherein X is a moiety PO₄or S═PO₃; wherein R₁ and R₂ are the same or different nucleosides;wherein R₁ and R₂ are selected from the group consisting ofβ-D-deoxyfluorouridine, α-L-deoxyfluorouridine, β-L-deoxyfluorouridine,α-L-deoxycytidine, β-L-deoxycytidine, β-L-deoxyuridine,β-L-deoxyguanosine, β-L-deoxyadenosine, α-L-deoxyadenosine andnitrobenzylthionosine; and wherein R₁ and R₂ are attached to X through—OH groups.
 2. The pharmaceutical composition according to claim 1wherein R₁ is β-D-deoxyfluorouridine.
 3. The pharmaceutical compositionaccording to claim 1 wherein R₁ is α-L-deoxyfluorouridine.
 4. Thepharmaceutical composition according to claim 1 wherein R₁ isβ-L-deoxyfluorouridine.
 5. The pharmaceutical composition according toclaim 1 wherein R₁ is α-L-deoxycytidine.
 6. The pharmaceuticalcomposition according to claim 1 wherein R₁ is β-L-deoxycytidine.
 7. Thepharmaceutical composition according to claim 1 wherein R₁ isβ-L-deoxyguanosine.
 8. The pharmaceutical composition according to claim1 wherein R₁ is β-L-deoxyguanosine.
 9. The pharmaceutical compositionaccording to claim 1 wherein R₁ is β-L-deoxyadenosine.
 10. Thepharmaceutical composition according to claim 1 wherein R₁ isα-L-deoxyadenosine.
 11. The pharmaceutical composition according toclaim 1 wherein R₁ is nitrobenzylthionosine.
 12. A pharmaceuticalcomposition of a nucleoside dimer comprising: β-D-deoxyfluorouridine,β-L-deoxyadenosine, and a suitable moiety for linking the two saidnucleosides selected from the group consisting of PO₄ or S═PO₃.